The K⁺ and NO₃⁻ Interaction Mediated by NITRATE TRANSPORTER1.1 Ensures Better Plant Growth under K⁺-Limiting Conditions

NITRATE TRANSPORTER 1.1 favors the uptake and allocation of K⁺, which respectively depends on its coordination with the K⁺ channels/transporters in the epidermis, cortex and central vasculature of roots.

Abstract

K⁺ and NO₃⁻ are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO₃⁻ and K⁺ in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K⁺ uptake and root-to-shoot allocation, and were characterized by growth arrest under K⁺-limiting conditions. The K⁺ uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K⁺ contents in nrt1.1-1/K⁺ transporter1, nrt1.1-1/high-affinity K⁺ transporter5-3, nrt1.1-1/K⁺ uptake permease7, and nrt1.1-1/stelar K⁺ outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K⁺ channels/transporters located in the root epidermis–cortex and central vasculature. Further study revealed that these K⁺ uptake-related interactions are dependent on an H⁺-consuming mechanism associated with the H⁺/NO₃⁻ symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis–cortex and central vasculature are coordinated with K⁺ channels/transporters to improve K⁺ uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K⁺-limiting conditions.

Potassium (K) is an essential element for plant growth and development and contributes to determining the yield and quality of crops in agriculture production (Wang and Wu, 2013). However, the concentrations of soluble K⁺ in most soils are relatively low, which often limits plant growth (Maathuis, 2009). Although crop production can be increased by applying large amounts of potassic fertilizers to agricultural fields, only approximately one-half of the applied fertilizers is available to plants; the remainder accumulates as residues in soils, consequently leading to environmental contamination (Meena et al., 2016). Therefore, there is a pressing need to gain a more complete understanding of the molecular mechanisms underlying K⁺ transport and regulation in order to enhance the K⁺ utilization efficiency of plants. Accordingly, in the past few decades, researchers have focused on identifying K⁺ channels and transporters in plants, as well as the mechanisms underlying their regulation.

In Arabidopsis (Arabidopsis thaliana), 71 K⁺ channels and transporters have been identified and categorized into three channel (Shaker, Tandem-Pore K⁺, and K_ir-like) and three transporter (K⁺ uptake permeases [KT/HAK/KUP], High-affinity K⁺ transporters [HKT], and cation/proton antiporter [CPA]) families (Wang and Wu, 2010). Among these, the shaker inward K⁺ channel K⁺ TRANSPORTER1 (AKT1) and the KT/HAK/KUP K⁺ transporter HIGH-AFFINITY K⁺ TRANSPORTER5 (HAK5) have been characterized as the two major components that contribute to K⁺ uptake in roots, although they have been found to operate at different K⁺ levels (Pyo et al., 2010; Wang and Wu, 2013). AKT1 functions in plant K⁺ uptake over a wide range of K⁺ concentrations, whereas HAK5 shows high-affinity K⁺ transport activity (Gierth et al., 2005). Following its uptake into root epidermal cells, K⁺ is distributed to different plant organs or tissues. The Arabidopsis shaker-like outward-rectifying K⁺ channel STELAR K⁺ OUTWARD RECTIFIER (SKOR), the expression of which was first identified in stelar tissues, has been shown to facilitate K⁺ secretion into xylem sap, which is a critical step in long-distance K⁺ transport from roots to shoots (Gaymard et al., 1998). Recently, K⁺ UPTAKE PERMEASE7 (KUP7), a member of the KT/HAK/KUP family, was functionally characterized as a K⁺ transporter participating in both root K⁺ uptake and root-to-shoot K⁺ allocation, particularly under K⁺-limiting conditions (Han et al., 2016). However, the uptake affinity for K⁺ has been found to be considerably lower in KUP7 than in HAK5 (Wang and Wu, 2017).

In addition to the aforementioned K⁺ channels and transporters, other mineral elements, including Na⁺, Ca²⁺, and N, are known to have pronounced effects on K⁺ nutrition in plants. Given that N is the nutrient that is required in the greatest quantity by most plants and is the most widely used fertilizer nutrient in crop production, the relationships between N and K have long been investigated (Fageria and Baligar, 2005; Wang and Wu, 2013; Meng et al., 2016; Shin, 2017). Since the 1960s, physiological studies have revealed a close relationship between NO₃⁻ and K⁺ with regard to uptake and translocation (Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985; Drechsler et al., 2015). However, the coordination between these two nutrients in plant transport pathways remains to be extensively studied at the molecular level. We hypothesized that transporters involved in the transference of NO₃⁻ across cell membranes may play a role in controlling K⁺ nutrition in plants. Recently, NITRATE TRANSPORTER1.5 (NRT1.5), a member of the nitrate transporter1/peptide transporter family (NPF), initially identified as a pH-dependent bidirectional NO₃⁻ transporter (Lin et al., 2008), was shown to be involved in the control of K⁺ allocation in plants (Drechsler et al., 2015; Li et al., 2017; Du et al., 2019). Nevertheless, it was subsequently established that this function was merely associated with its role as a proton-coupled H⁺/K⁺ antiporter for K⁺ loading into the xylem (Li et al., 2017; Du et al., 2019), which is not associated with the transport of NO₃⁻. In this study, we showed that the loss of another nitrate transporter1 member, NRT1.1/NPF6.3, in nrt1.1 mutants led to the development of a more pronounced K⁺-deficiency phenotype under conditions of low-K⁺ stress. Further physiological and genetic evidence revealed that both the uptake and root-to-shoot allocation of K⁺ in plants require NRT1.1. However, NRT1.1 acts as a coordinator rather than a K⁺ channel/transporter in K⁺ uptake and root-to-shoot allocation, which could depend on its NO₃⁻-related transport activity. Our findings highlight the significance of nutrients and nutrient interactions in ensuring plant growth, and indicate that the modification of NRT1.1 homolog activity in crops using biological engineering techniques might be a promising approach that could simultaneously contribute to enhancing the utilization efficiencies of K and N fertilizers in agricultural production.

RESULTS

NRT1.1 Promotes Better Growth under K⁺-Limiting Stress Conditions

In Arabidopsis, six nitrate transporters, NRT1.1, NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5, have been found to play roles in root NO₃⁻ uptake (Wang et al., 2012; Léran et al., 2014; Lezhneva et al., 2014; Ye et al., 2019). In this study, we found that when cultured on growth medium containing 6 mm of NO₃⁻, the two NRT1.1 knockout mutants nrt1.1-1 and chl1-5 exhibited considerably greater leaf senescence compared to the wild-type plants (Col-0) after 8 d of low-K⁺ treatment (0.05 mm K⁺), whereas these three plant lines did not show distinguishable phenotypes when grown in the presence of sufficient K⁺ (2 mm K⁺; Fig. 1A). The low-K⁺-induced leaf senescence in the NRT1.1-knockout mutants was confirmed by measuring leaf chlorophyll contents (Fig. 1B). Given that a reduction in chlorophyll content would affect the photosynthesis of plants, we also evaluated the maximum photochemical efficiency of PSII in the dark-adapted state (F_v/F_m) and the photosynthetic electron transport rate (ETR) as indicators of the photosynthetic reactions in leaves. We accordingly found that under low-K⁺ conditions, F_v/F_m in the leaves of nrt1.1-1 and chl1-5 plants was almost completely abolished (Fig. 1C), and that there was also an apparent reduction in the ETR compared with that in Col-0 plants (Fig. 1D). These differences were reflected by the fact that the nrt1.1 knockout mutants had both shorter roots and a lower biomass compared with Col-0 plants after low-K⁺ treatment (Fig. 1, E and F; Supplemental Fig. S1). In addition, we examined a third NRT1.1-null mutant, chl1-6, the fresh weight and root elongation of which were also significantly reduced compared with those of the corresponding Landsberg erecta (Ler) wild type under low-K⁺ conditions (Supplemental Fig. S2, A and B). All these observations show that NRT1.1 is required for tolerance to K⁺ deficiency. NRT1.1 also functions as an NO₃⁻ sensor, which is independent of its role in NO₃⁻ uptake (Ho et al., 2009). The NRT1.1 P492L point mutant chl1-9 is defective in NO₃⁻ uptake but has a normal NO₃⁻-sensing function (Ho et al., 2009). Consequently, we examined the chl1-9 mutant to clarify whether it is the NO₃⁻-uptake activity or the NO₃⁻-sensing function that is involved in the tolerance to K⁺ deficiency. If the NO₃⁻-sensing function of NRT1.1 was required for tolerance to K⁺ deficiency, the chl1-9 mutant would have a phenotype similar to that of the wild-type plant but not the NRT1.1 knockout mutant chl1-5. However, we found that the chl1-9 mutant had growth inhibition similar to that observed in the chl1-5 mutant under low-K⁺ conditions (Supplemental Fig. S3). The result indicates that NO₃⁻-uptake activity, rather than the NO₃⁻-sensing function, is probably responsible for NRT1.1-mediated tolerance to K⁺ deficiency.

Figure 1.

Loss of NRT1.1 function in an Arabidopsis nrt1.1 mutant leads to hypersensitivity to low-K⁺ stress. A, Photographs of plant phenotypes. B, Chlorophyll content of leaves. C, Maximal quantum efficiency of PSII. D, ETR. E, Root elongation. F, Biomass of plants. Four-day-old seedlings of Col-0, nrt1.1-1, and chl1-5 were transferred to an agar medium containing 2 or 0.05 mm K⁺, as described in “Materials and Methods”. Analyses were performed 8 d after seedling transfer. Bars represent the mean ± sd (n = 5). Different letters above the bars in B, E, and F indicate significant difference within the same treatment using LSD test (P < 0.05) and asterisks indicate a significant genotype by treatment interaction using two-way ANOVA (***P < 0.001). Asterisks in D show significant differences compared with Col-0 using two-tailed Student’s t-test (*P < 0.05 and ***P < 0.001). FW, Fresh weight; ns, nonsignificant.

We subsequently determined the roles of the other five NRTs in plant growth responses to low-K⁺ stress. Given that NRT2.1, NRT2.2, NRT2.4, and NRT2.5 act as high-affinity NO₃⁻ transporters, we examined the growth of the null mutants of these NRTs in a growth medium containing 0.2 mm of NO₃⁻. However, we found that the fresh weight and root elongation of the nrt2.1, nrt2.2, nrt2.4, and nrt2.5 mutants were similar to those of the corresponding wild-type plants in response to both low- and sufficient-K⁺ treatments (Supplemental Fig. S4, A–D and F–I). Similarly, although NRT1.2 is a low-affinity NO₃⁻ transporter (Huang et al., 1999), the nrt1.2 mutant showed fresh weight and root elongation comparable to those of wild-type plants when grown on both low- and sufficient-K⁺ growth medium containing 6 mm of NO₃⁻ (Supplemental Fig. S4, E and J). These results indicate that NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5 do not function similarly to NRT1.1 with respect to conferring tolerance to K⁺ deficiency.

NRT1.1 Improves K⁺ Nutrition in Plants, and K⁺-Limiting Conditions Stimulate the Activity of NRT1.1 in Roots

We next sought to determine the mechanism by which NRT1.1 confers tolerance to K⁺ deficiency in plants cultured on growth medium containing a sufficient supply of NO₃⁻. In response to both sufficient- and low-K⁺ treatments, K⁺ levels were found to be significantly lower in nrt1.1-1, chl1-5, and chl1-6 mutants than in the corresponding wild-type plants (Fig. 2, A and B; Supplemental Fig. S2, C and D), indicating that a lack of NRT1.1 activity perturbs K⁺ nutrition in plants. However, when the plants were grown in a medium containing low NO₃⁻ (0.2 mm), we detected no significant differences in either the growth or K⁺ levels of NRT1.1-null mutants and wild-type plants in both sufficient- and low-K⁺ treatments (Supplemental Fig. S5, A–D). Accordingly, these results indicate that the role of NRT1.1 in improving K⁺ nutrition in plants is dependent on a sufficient supply of NO₃⁻. To provide further evidence in support of the role of NRT1.1 in K⁺ nutrition, we measured K⁺ levels in pNRT1.1::NRT1.1-GFP transgenic plants, which are in a chl1-5 mutant background (Krouk et al., 2010). In response to both sufficient- and low-K⁺ treatments, we observed that the pNRT1.1::NRT1.1-GFP plants contained K⁺ levels higher than those of the chl1-5 mutants but similar to those in Col-0 plants (Fig. 2C). Phenotypic analysis revealed that complementation with NRT1.1 conferred tolerance to K⁺ deficiency in transgenic plants, as their chlorophyll content, root elongation, and fresh weight were comparable to those of Col-0 plants (Fig. 2D; Supplemental Fig. S6).

Figure 2.

NRT1.1 is required to maintain K⁺ nutrition in Arabidopsis. A and B, Shoot and root K⁺ contents of Col-0, nrt1.1-1, and chl1-5 plants. C, Shoot and root K⁺ contents of Col-0, chl1-5, and pNRT1.1::NRT1.1-GFP plants. Bars represent the mean ± sd (n = 5). Asterisks indicate significant differences between genotypes using two-tailed Student’s t-test (*P < 0.05 and **P < 0.01). D, Photographs of the phenotypes of Col-0, chl1-5, and pNRT1.1::NRT1.1-GFP plants. Four-day-old seedlings were treated as described in the Figure 1 legend. E, Phenotypes of grafted plants. F and G, K⁺ contents of grafted plants. The grafted seedlings were transferred to an agar medium containing 2.0 or 0.05 mm K⁺. Analyses were performed 8 d after seedling transfer. Bars represent the mean ± sd (n = 5). Different letters above bars indicate significant differences within the same treatment using LSD test (P < 0.05). DW, Dry weight; ns, nonsignificant.

The role of root NRT1.1 in improving plant K⁺ nutrition prompted us to investigate the mechanism by which the activity of NRT1.1 in roots responds to low-K⁺ stress. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed that low-K⁺ treatment clearly increased the expression of NRT1.1 in roots (Fig. 3A), whereas expression of the other five NO₃⁻ uptake transporter genes was either reduced (NRT2.1; Supplemental Fig. S7A) or not significantly affected (NRT1.2, NRT2.2, NRT2.4, and NRT2.5; Supplemental Fig. S7, B–E), thereby indicating that low-K⁺ stress might have a specific effect on the activity of NRT1.1. Consistent with the observed gene expression patterns, we detected an increase in the expression of the NRT1.1-GFP protein (as indicated by GFP-associated fluorescence) in the roots of pNRT1.1::NRT1.1-GFP transgenic plants in response to low-K⁺ treatment as compared to that in plants in sufficient-K⁺ conditions (Fig. 3B). These findings indicated that the activity of NRT1.1 could be upregulated in response to low-K⁺ stress, and therefore, we analyzed the rate of net NO₃⁻ fluxes at the surface of the maturation, elongation, and meristematic zones of Col-0 roots using noninvasive microtest technology. We accordingly found that in plants precultured on low-K⁺ medium, all three root zones showed a higher rate of net NO₃⁻ influx compared with plants precultured with sufficient-K⁺ medium in the same testing medium (Fig. 3C). These observations thus provided direct evidence that low-K⁺ stress stimulates NO₃⁻ uptake activity, which is presumably associated with an upregulation of NRT1.1 due to low-K⁺ stress.

Figure 3.

Low-K⁺ stress stimulates NRT1.1 activity in roots. A, Relative expression of NRT1.1 in the roots of Arabidopsis Col-0 plants. The relative expression levels were normalized to the levels of UBQ10. Bars represent the mean ± sd (n = 5). Asterisks indicate significant differences determined using two-tailed Student’s t-tests (**P < 0.01). B, NRT1.1-GFP expression in pNRT1.1::NRT1.1-GFP transgenic plants. Scale bars = 100 µm. C, Average values of net NO₃⁻ fluxes in the meristematic, elongation, and maturation zones of Col-0 roots. Four-day-old seedlings were precultured in an agar medium containing 2 or 0.05 mm K⁺. Analyses were performed 5 d after seedling transfer. Negative values in C indicate a net influx. Bars represent the mean ± sd (n = 5). Different letters above bars indicate significant differences using LSD test (P < 0.05).

Both the Uptake and Root-to-Shoot Allocation of K⁺ Require the Coordination of NRT1.1

We went on to investigate how NRT1.1 enhances K⁺ nutrition. Given that NRT1.1 is expressed in both roots and shoots (Guo et al., 2001), we generated homografted (Col-0 scion/Col-0 stock and nrt1.1-1 scion/nrt1.1-1 stock) and heterografted (Col-0 scion/nrt1.1-1 stock and nrt1.1-1 scion/Col-0 stock) plants to clarify whether either the roots or the shoots, or both, are sites of NRT1.1 activity associated with an enhancement of K⁺ nutrition in plants. After 8 d of growth, the leaves of Col-0/nrt1.1-1 and nrt1.1-1/nrt1.1-1 plants grown on low-K⁺ medium had developed a severe chlorotic phenotype, whereas no comparable chlorotic symptoms were evident on nrt1.1-1/Col-0 and Col-0/Col-0 plants (Fig. 2E). These observations indicated that the absence of the root-associated activity of NRT1.1 was primarily responsible for the susceptibility to chlorosis. This assumption was further supported by the observation that K⁺ levels were clearly reduced in the shoots of plants grafted with nrt1.1-1 root stock in low-K⁺ treatment (Fig. 2G). Furthermore, in plants exposed to sufficient K⁺, we found that K⁺ levels in both shoots and roots were significantly lower in the Col-0/nrt1.1-1 and nrt1.1-1/nrt1.1-1 plants than in the plants grafted with Col-0 rootstock (Fig. 2F). Collectively, these results indicate that only the root-associated activity of NRT1.1 contributes to an enhancement of K⁺ nutrition in plants.

In subsequent analyses, we therefore focused our attention on the root activity of NRT1.1. We initially evaluated K⁺ uptake by roots using Rb⁺, which is the analog most similar to K⁺. As shown in Fig. 4A, the rates of root Rb⁺ uptake in nrt1.1-1 and chl1-5 mutants were considerably lower than those in Col-0 plants in either 2 or 0.05 mm Rb⁺ medium, indicating that loss of NRT1.1 function might disturb the uptake of K⁺ by roots. To further evaluate the contribution of NRT1.1 to root K⁺ uptake, we measured root K⁺ flux in the aforementioned three plant lines using noninvasive microtest technology and found that in the elongation and maturation zones, the rates of net K⁺ influx in nrt1.1-1 and chl1-5 mutants were <50% of those obtained in the Col-0 plants in both 2 and 0.05 mm of K⁺ testing media. In contrast, we detected no significant difference among plant lines with respect to net K⁺ influx in the meristematic zone (Fig. 4, B and C). These results indicate that the elongation and maturation zones, although not the meristematic zone, are the target regions of NRT1.1 associated with K⁺ uptake by root cells.

Figure 4.

NRT1.1 is required for both uptake and root-to-shoot allocation of K⁺ in Arabidopsis. A, Rate of Rb⁺ uptake in roots. Four-day-old seedlings were transferred to an agar medium containing 2 or 0.05 mm Rb⁺ for 12 h, as described in “Materials and Methods”. Bars represent the mean ± sd (n = 5). FW, Fresh weight. B and C, Net K⁺ fluxes in roots. Four-day-old seedlings were precultured in an agar medium containing 2 or 0.05 mm K⁺ for 3 d and then used to measure net K⁺ fluxes in the meristematic, elongation, and maturation zones under 2 or 0.05 mm K⁺ treatment. Negative values indicate a net influx; bars represent the mean ± sd (n = 5). D, Proportions of K⁺ distributed in the shoots and roots. Four-day-old seedlings were treated as described in the Figure 1 legend. Bars represent the mean ± sd (n = 5). Asterisks indicate significant differences compared with Col-0 plantsusing two-tailed Student’s t-test (*P < 0.05 and **P < 0.01). ns, Nonsignificant.

Interestingly, our determinations of the proportions of K⁺ distributed in the roots and shoots of plants revealed that when grown in low-K⁺ medium, both nrt1.1-1 and chl1-5 mutants were characterized by a lower proportion of K⁺ distributed in the shoots but a higher proportion distributed in the roots compared with Col-0 plants (Fig. 4D). In contrast, we detected no significant differences in the proportions of K⁺ distributed in the shoots and roots of Col-0 plants and nrt1.1 knockout mutants when these were grown on sufficient-K⁺ medium. Based on these observations, we can deduce that NRT1.1 also plays a distinct role in the root-to-shoot allocation of K⁺, although only under K⁺-limiting conditions.

Action of NRT1.1 in Favoring the Uptake and Root-to-Shoot Allocation of K⁺ Depends on Its Specific Expression in the Epidermis-Cortex and Central Vasculature, Respectively, of Roots

As NRT1.1 is expressed in both the epidermis-cortex and central vasculature of roots (Huang et al., 1996; Remans et al., 2006), we sought to determine whether these expression patterns are correlated with a dual role of NRT1.1 in coordinating the uptake and root-to-shoot allocation of K⁺. To this end, we generated transgenic plants expressing NRT1.1 under control of the promoters of SULFATE TRANSPORTER1;2 (Sultr1;2) and PHOSPHATE1 (PHO1) in the nrt1.1-1 mutant background. Sultr1;2 is expressed in the root epidermis and cortex (Yoshimoto et al., 2002), whereas PHO1 is expressed in the root central vasculature (Hamburger et al., 2002; Wege and Poirier, 2014). Therefore, use of these two promoters to drive NRT1.1 expression in transgenic plants could ensure NRT1.1 expression in the epidermis-cortex and central vasculature of roots. The transcripts of NRT1.1 were partially recovered in pSultr1;2::NRT1.1 and pPHO1::NRT1.1 transgenic lines compared with the nrt1.1-1 mutant, although the transcript levels were not well matched with those in wild-type plants (Supplemental Fig. S8). A comparison of K⁺ levels in pSultr1;2::NRT1.1 transgenic lines with those in Col-0 and nrt1.1-1 plants revealed a partial recovery of K⁺ to wild-type levels in the plants subjected to both sufficient- and low-K⁺ treatments (Fig. 5, A and B). However, determination of the proportions of K⁺ distributed in the roots and shoots revealed that pSultr1;2::NRT1.1 complementation in nrt1.1-1 plants did not improve the root-to-shoot allocation of K⁺ (Fig. 5C). Accordingly, these observations tend to indicate that expression of NRT1.1 only in the epidermis and cortex might be responsible for improving the K⁺ uptake.

Figure 5.

NRT1.1 expression driven by Sultr1;2 and PHO1 promoters partially rescued the uptake and root-to-shoot allocation, respectively, of K⁺ in nrt1.1 mutants. A to C, K⁺ content (A and B) and distribution (C) in Col-0, nrt1.1-1, and two independent pSultr1;2::NRT1.1-transformed nrt1.1-1 lines (pSultr1;2::NRT1.1-1 and pSultr1;2::NRT1.1-2). D to F, K⁺ content (D and E) and K⁺ distribution (F) in Col-0, nrt1.1-1, and two independent pPHO1::NRT1.1-transformed nrt1.1-1 lines (pPHO1::NRT1.1-1 and pSultr1;2::NRT1.1-2). Four-day-old seedlings were treated as described in the Figure 1 legend. Bars represent the mean ± sd (n = 5). Different letters above bars indicate significant differences using LSD test (P < 0.05). DW, Dry weight.

Measurement of K⁺ levels in the two independent pPHO1::NRT1.1 transgenic lines also revealed a partial recovery of K⁺ to Col-0 levels in plants subjected to both K⁺ treatments, with the recovery being more evident in the shoots (Fig. 5, D and E). Furthermore, examination of the proportions of K⁺ distributed in the roots and shoots revealed that the complementation of pPHO1::NRT1.1 in nrt1.1-1 plants increased K⁺ transport from roots to shoots (Fig. 5F), thereby indicating that the expression of NRT1.1 in the central vasculature of roots is responsible for supporting the root-to-shoot allocation of K⁺.

Action of NRT1.1 in Favoring the Uptake and Root-to-Shoot Allocation of K⁺ Requires the Collaboration of K⁺ Channels/Transporters in the Epidermis-Cortex and Central Vasculature, Respectively, of Roots

We subsequently investigated the mechanism by which the expression of NRT1.1 in the epidermis-cortex and central vasculature coordinates the uptake and root-to-shoot allocation, respectively, of K⁺. In addition to its role in mediating nitrate transport across the plasmalemma, NRT1.1 has also been found to function as a signaling component in regulating the expression of numerous genes in plants (Ho et al., 2009; Bouguyon et al., 2015; Wang et al., 2018). Therefore, we conducted RNA-sequencing analysis to compare differences in the whole-genome gene expression profiles of Col-0 plants and nrt1.1 mutants in response to different K⁺ treatments. However, among the genes associated with K⁺ uptake and trafficking, only the expression of SKOR, which mediates K⁺ loading into the xylem (Gaymard et al., 1998; Drechsler et al., 2015), was inhibited by the lack of NRT1.1 in both sufficient- and low-K⁺ treatments, whereas that of other genes was either increased or not significantly affected (Supplemental Fig. S9A). Given that these results cannot account for the observed reduction in root K⁺ uptake due to the loss of NRT1.1 function, we also investigated whether, similar to NRT1.5, NRT1.1 has K⁺ transport activity (Li et al., 2017). In order to functionally examine the role of NRT1.1 in K⁺ transport activity in a heterologous expression system, we used the R5421 strain of yeast (Saccharomyces cerevisiae), which lacks the main K⁺ uptake systems (trk1Δ, trk2Δ; Gaber et al., 1988; Han et al., 2016). However, we found that NRT1.1 was unable to complement the retardation of growth in R5421 in response to K⁺ deficiency (Supplemental Fig. S9B).

We therefore assumed that the action of NRT1.1 in improving plant K⁺ nutrition might require the collaboration of K⁺ channels/transporters. To test this hypothesis, we generated nrt1.1-1/akt1, nrt1.1-1/hak5-3, nrt1.1-1/skor-2, and nrt1.1-1/kup7 double mutants by crossing the NRT1.1-null mutant nrt1.1-1 with akt1, hak5-3, skor-2, and kup7 mutants, and measured K⁺ levels in these plants. AKT1, HAK5, and KUP7 are the three characterized components that contribute to K⁺ uptake in Arabidopsis roots, among which only the latter two make a significant contribution to K⁺ uptake under K⁺-limiting conditions (Lagarde et al., 1996; Gierth et al., 2005; Han et al., 2016). In addition, KUP7, along with SKOR channels, has also been found to be involved in K⁺ loading into the xylem (Han et al., 2016). Two-way ANOVA revealed that in response to both 2 and 0.05 mm K⁺ treatments, differences in K⁺ levels in the shoots and roots of akt1 and nrt1.1-1/akt1 mutants were significantly less than those between Col-0 and nrt1.1-1 (Fig. 6, A and B), indicating that the reduced K⁺ levels attributable to loss of NRT1.1 are associated with the action of AKT1. Similarly, two-way ANOVA based on the data obtained from Col-0, nrt1.1-1, hak5-3, nrt1.1-1/hak5-3, kup7, and nrt1.1-1/kup7 plants revealed that reduced K⁺ levels due to the loss of NRT1.1 were also associated with HAK5 and KUP7 in low-K⁺ medium (Fig. 6, C and E). In addition, the findings from biomass analysis indicated that under low-K⁺ conditions, growth arrest due to loss of NRT1.1 was also associated with the aforementioned K⁺ channels/transporters (Supplemental Fig. S10). These results indicate that NRT1.1 should interplay with K⁺ uptake channels/transporters in the root epidermis-cortex to improve plant K⁺ nutrition. We next determined whether KUP7 and SKOR are required for NRT1.1 activity favoring the root-to-shoot allocation of K⁺ under low-K⁺ conditions. Two-way ANOVA revealed that differences in proportion of K⁺ transferred from roots to shoots between either skor-2 and nrt1.1-1/skor-2 or kup7 and nrt1.1-1/kup7 plants were significantly lower than differences between Col-0 and nrt1.1-1 plants (Fig. 6, F and H), thereby indicating that the action of NRT1.1 in favoring root-to-shoot allocation of K⁺ requires the collaboration of K⁺ channels/transporters that mediate K⁺ loading into the xylem.

Figure 6.

Physiological interactions between NRT1.1 and K⁺ channels/transporters. A and B, K⁺ content in Col-0, nrt1.1-1, akt1, and nrt1.1-1/akt1 plants. C and D, K⁺ content in Col-0, nrt1.1-1, hak5-3, and nrt1.1-1/hak5-3 plants. E and F, K⁺ content (E) and distribution (F) in Col-0, nrt1.1-1, kup7, and nrt1.1-1/kup7 plants. G and H, K⁺ content (G) and distribution (H) in Col-0, nrt1.1-1, skor-2, and nrt1.1-1/skor-2 plants. Four-day-old seedlings were treated as described in the Figure 1 legend. Bars represent the mean ± sd (n = 5). Different letters above bars indicate significant differences using LSD test (P < 0.05). Asterisks indicate a significant genotype by genotype interaction using two-way ANOVA (*P < 0.05 and **P < 0.01). DW, Dry weight; ns, nonsignificant.

The NRT1.1-Improved K⁺ Nutrition Is Associated with the Prevention of Rhizosphere Acidification

Uptake of K⁺ across the plasmalemma of the root cortex cells is coupled to H⁺-ATPase-mediated H⁺ efflux, which leads to hyperpolarization of the plasmalemma (Wang and Wu, 2013). In this regard, it is assumed that a factor that promotes an increase in the activity of H⁺-ATPase would contribute to enhancing the uptake of K⁺. The optimum pH for plasmalemma H⁺-ATPase activity in plant roots is ∼6.2 to 6.5 (De Michelis and Spanswick, 1986; Cowan et al., 1993; Zhu et al., 2009), and lowering the pH of the growth medium has been found to result in a marked reduction in K⁺ uptake by roots (Jacobson et al., 1957; Rains et al., 1964; Zsoldos and Erdei, 1981; Bergback and Borg, 1989). In this study, we also investigated the effect of different pH on plant K⁺ contents, and detected the highest K⁺ contents in plants grown on medium at pH 6 to 6.5, whereas values either above or below this range reduced the K⁺ contents in plants (Supplemental Fig. S11). Thus, in the absence of a mechanism to eliminate H⁺, acidification resulting from H⁺-ATPase-mediated H⁺ efflux during K⁺ uptake may in turn prove unfavorable from the perspective of K⁺ nutrition. Recently, it has been demonstrated that NRT1.1 plays an important role in preventing acidification of the rhizosphere (Fang et al., 2016; Zhu et al., 2019), which is associated with its H⁺/NO₃⁻ symport activity (Tsay et al., 1993). Similarly, in this study, we found that NRT1.1 is required to prevent rhizospheric acidification under both sufficient- and low-K⁺ conditions (Fig. 7, A and B). We therefore investigated whether NRT1.1-improved K⁺ nutrition is associated with the prevention of rhizosphere acidification by buffering the pH of growth media. In comparison with the results obtained in treatments without MES, the presence of MES, which substantially minimizes the differences in pH of the rooting media of Col-0 and the nrt1.1-1 and chl1-5 mutants (Fig. 7, A and B), clearly improved the growth of the two NRT1.1 knockout mutants under low-K⁺ conditions (Fig. 7C; Supplemental Fig. S12). Furthermore, the presence of MES also clearly elevated K⁺ levels and the Rb⁺ uptake rate in two nrt1.1 mutants under both sufficient- and low-K⁺ conditions (Fig. 7, D–G; Supplemental Fig. S13), thereby minimizing the differences between Col-0 plants and the nrt1.1 mutants. Similarly, we examined the pH buffering effects of Bis-Tris and found that this buffer also significantly increased K⁺ contents in the two nrt1.1 mutants under both sufficient- and low-K⁺ conditions, consequently reducing the differences between Col-0 plants and the nrt1.1 mutants (Supplemental Fig. S14). These results accordingly confirmed our speculation that the NRT1.1-mediated improvement of K⁺ nutrition is associated with the prevention of rhizosphere acidification.

Figure 7.

pH buffering of the growth medium enhances the K⁺ nutrition of Arabidopsis nrt1.1 mutants. A and B, pH of the growth medium with or without a pH buffer. C, Phenotypes of plants grown on pH-buffered medium. D to G, Shoot and root K⁺ contents of plants grown with or without pH buffer. Seedlings were transferred to an agar medium supplemented with 2 or 0.05 mm K⁺ and with or without MES (0.05% [w/v]). The initial pH of the growth medium was 6.5, and analyses were performed 10 d after seedling transfer. Bars represent the mean ± sd (n = 5). Asterisks show significant differences compared with Col-0 plants using two-tailed Student’s t-test (*P < 0.05 and **P < 0.01). DW, dry weight; ns, nonsignificant.

DISCUSSION

K⁺ and NO₃⁻ are the major forms of potassium and nitrogen absorbed by the roots of most terrestrial plants. In addition, they represent the most abundant inorganic cation and anion sources, respectively, in plant cells and are thus also the major forms of potassium and nitrogen transported within plants (Du et al., 2019). In this study, we revealed a close relationship between NO₃⁻ and K⁺ nutrition, which is mediated by the NO₃⁻ transporter NRT1.1, in the root tissues of Arabidopsis.

NRT1.1 was initially characterized as an NO₃⁻ transporter involved in root NO₃⁻ uptake from the growth medium (Tsay et al., 1993). Subsequently, it was demonstrated to be a bidirectional transporter (Remans et al., 2006) that functions as a component involved in xylem NO₃⁻ loading in the root stele, thereby contributing to the root-to-shoot allocation of NO₃⁻ (Léran et al., 2013). Further studies have revealed that NRT1.1 is characterized by a number of additional functions, including regulation of another NO₃⁻ transporter, NRT2.1 (Ho et al., 2009); auxin transport (Krouk et al., 2010); and regulation of tolerance to NH₄⁺ toxicity (Jian et al., 2018). The latter functions of NRT1.1 are, nevertheless, independent of its NO₃⁻ transport activity, given that an absence of NO₃⁻ in the growth medium does not affect them. However, our finding that neither the roots nor shoots of wild-type plants show higher K⁺ levels than those of nrt1.1 knockout mutants when NH₄⁺ is the sole nitrogen source indicates that NO₃⁻ transport across the plasmalemma is probably a prerequisite for the NRT1.1-mediated improvement of K⁺ nutrition in plants (Supplemental Fig. S15, C and D). It has long been recognized that the processes of NO₃⁻ uptake and transport need K⁺ to maintain electroneutrality (Marschner, 1995; Anjana and Iqbal, 2007; Delaire et al., 2014; Li et al., 2017), and this interdependency of NO₃⁻ and K⁺ may provide an explanation for the above notion that NRT1.1-improved K⁺ nutrition is dependent on NO₃⁻-uptake activity. Theoretically, it is assumed that the NO₃⁻ transport-associated enhancement of K⁺ nutrition in plants follows a general mechanism. However, we found that other nitrate uptake NRTs do not function similarly to NRT1.1 when coordinating K⁺ nutrition in plants (Supplemental Fig. S16). In this regard, previous studies have found that NRT1.1 contributes to >70% of the total root NO₃⁻ uptake in plants grown on NO₃⁻-sufficient medium (Huang et al., 1996; Wang et al., 1998), thereby indicating that the other NRTs (i.e. NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5) might be responsible for <30% of NO₃⁻ uptake. Therefore, the insufficient NO₃⁻ transport of these five NRTs might explain why none play an evident role in enhancing K⁺ nutrition in plants. This also suggests that the action of NRT1.1 to improve K⁺ nutrition might require a higher NO₃⁻ supply to ensure sufficient NO₃⁻ transport. This assumption is supported by our observation that NRT1.1-null mutants do not have distinguishable growth phenotypes or differences in K⁺ levels compared to wild-type plants when grown on low-NO₃⁻ growth medium (0.2 mm NO₃⁻; Supplemental Fig. S5). Interestingly, improvement of root uptake and root-to-shoot allocation of K⁺ by NRT1.1 in Arabidopsis is dependent on the nature of NRT1.1 expression patterns in the root epidermis-cortex and central vasculature, respectively (Fig. 5), and it is thus necessary to determine the underlying mechanism.

Complementation studies using yeast trk1Δ trk2Δ mutants failed to provide any evidence that NRT1.1 functions as a K⁺ transporter (Supplemental Fig. S9B). Although one plausible explanation in this regard is that NRT1.1 lacks K⁺ transport activity, it is known that the findings of functional characterizations of plant transporters using heterologous expression systems are sometimes inconclusive, which is conceivably associated with the incompatibilities inherent in heterologous systems (Dreyer et al., 1999). Given this limitation, we cannot completely rule out the possibility that NRT1.1 has K⁺ transport activity based on the use of yeast expression systems. Nevertheless, in this study, we provide evidence that NRT1.1 in the root epidermis-cortex and central vasculature interplay with K⁺ channels/transporters to improve K⁺ uptake and root-to-shoot allocation, respectively (Fig. 6). It has been suggested that H⁺-coupled K⁺ uptake is a type of K⁺ uptake used by some K⁺ channels/transporters. Although this mechanism needs to be further confirmed based on biophysical analyses, we agree that H⁺-coupled K⁺ transport is one of the mechanisms for K⁺ uptake in plants, as some studies have shown that lowering the pH in the growth medium increases the rate of K⁺ uptake by roots (e.g. the study of Behl and Raschke, 1987). The findings of other studies, however, have provided conflicting evidence. For example, several studies have found that lowering the pH of the growth medium has the effect of reducing uptake of K⁺ by roots of wheat (Triticum aestivum; Zsoldos and Erdei, 1981), spruce (Picea sp.; Bergback and Borg, 1989), and barley (Hordeum vulgare; Rains et al., 1964). These observations accordingly indicate that certain other mechanisms are probably involved in the modulation of K⁺ uptake with respect to H⁺ (pH). One plausible mechanism in this regard is that H⁺ (pH) could affect K⁺ uptake by modulating the activity of H⁺-ATPase, given that the uptake of K⁺ by roots is electrochemically coupled to an H⁺-ATPase-mediated H⁺ efflux (Behl and Raschke, 1987; Wang and Wu, 2013). Consequently, to maintain a suitable rhizospheric pH and thereby preferable H⁺-ATPase activity for K⁺ uptake, an H⁺ elimination mechanism is necessary to balance the H⁺-ATPase-mediated H⁺ efflux during K⁺ uptake. Here, we demonstrated that the NRT1.1-mediated H⁺/NO₃⁻ symport across the plasmalemma represents such a mechanism that facilitates root K⁺ uptake, which is based on our observations that both the Rb⁺ uptake rate and the K⁺ contents and growth of nrt1.1 mutants were enhanced to levels comparable to those of Col-0 plants when cultivated on MES-buffered growth medium (Fig. 7, C–G; Supplemental Fig. S13). Accordingly, we speculate that NRT1.1-enhanced K⁺ uptake is mediated via an indirect route rather than by the direct K⁺ transport activity of NRT1.1, and thus, such action is probably coordinated by K⁺ channels/transporters in the root epidermis-cortex. This conjecture is supported by the findings of our two-way ANOVA based on K⁺ levels in nrt1.1/akt1, nrt1.1/hak5, and nrt1.1/kup7 double mutants and the corresponding single mutants and wild-type plants (Fig. 6, A–E; Supplemental Fig. S17A). As all the K⁺ channels/transporters we investigated can interact physiologically with NRT1.1 to improve K⁺ uptake, we suggest that these interactions could function in a nonspecific manner and might represent an underlying mechanism for maintaining electroneutrality during NO₃⁻ uptake (Marschner, 1995; Anjana and Iqbal, 2007; Delaire et al., 2014; Li et al., 2017). Although we provide no molecular evidence for direct protein-protein interactions in this regard, a common component, CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE23 (CIPK23), was previously shown to modulate the uptake activities of the K⁺ channel AKT1 and K⁺ transporter HAK5, as well as the affinity of the NO₃⁻ transporter NRT1.1, in several studies (Ho et al., 2009; Ragel et al., 2015; Wang et al., 2016; Sánchez-Barrena et al., 2020). Regulation of the uptake of NO₃⁻ and K⁺ by the same kinase leads us to suspect that the aforementioned interactions might be coordinated, or at least partially coordinated, at the molecular level. The role of the physiological interactions between NRT1.1 and K⁺ channels/transporters (e.g., SKOR and KUP7) in the root central vasculature in improving root-to-shoot allocation of K⁺ could also provide a means of meeting the requirement of electroneutrality during xylem NO₃⁻ loading mediated by NRT1.1. Unfortunately, in this study we were unable to clarify whether the interaction between K⁺ and NO₃⁻ is also associated with a pH modulation via the H⁺/NO₃⁻ symport activity of NRT1.1, owing to technical difficulties related to buffering pH fluctuations in the root central vasculature. However, it is worth noting that in addition to the aforementioned mechanism, transcript regulation by NRT1.1 might also play a role in improving the root-to-shoot allocation of K⁺, given that we found that expression of SKOR is positively regulated by NRT1.1 (Supplemental Fig. S9A). We also analyzed the distribution of K⁺ in the shoot and root tissues of nrt1.1/akt1 and nrt1.1/hak5 double mutants and the corresponding single mutants and wild-type plants, but we detected no physiological interaction between NRT1.1 and AKT1 or HAK5 with respect to enhancing the root-to-shoot allocation of K⁺, as revealed by two-way ANOVA (Supplemental Fig. S17, C and D). These findings indicate that K⁺ uptake channels/transporters might not be involved in the NRT1.1-coordinated root-to-shoot allocation of K⁺. Furthermore, our observations tended to indicate that the contribution of NRT1.1 to the root-to-shoot allocation of K⁺ is only sufficiently pronounced under conditions of K⁺ limitation (Fig. 4D). Our finding that efficient functioning of NRT1.1 in the uptake and root-to-shoot allocation of K⁺ requires a sufficient concentration of NO₃⁻ (Supplemental Fig. S5E) indicated that NRT1.1 may require a higher ratio of NO₃⁻ to K⁺ transport across the cell plasmalemma to make an appreciable contribution to the root-to-shoot allocation of K⁺ in plants. Given that the K⁺ in plants can be readily allocated to shoots following its uptake into roots from the growth medium (De Boer, 1999), the amount of K⁺ loaded into the root xylem needs to be considerably higher under K⁺-sufficient conditions, which results in a lower ratio of NO₃⁻ to K⁺ transport across the cell plasmalemma in the root central vasculature. This might explain why the contribution of NRT1.1 to the root-to-shoot allocation of K⁺ is not particularly marked under K⁺-sufficient conditions.

It is also worth noting that NRT1.1 plays a role in activating gene expression of the MADS-box transcription factor ARABIDOPSIS NITRATE REGULATED 1 (ANR1) to promote lateral root elongation in response to local NO₃⁻ supply (Remans et al., 2006), and thus, in this study, we also examined the possible association between NRT1.1 and root morphology. However, we failed to detect any significant differences in either number or average length of lateral roots in Col-0 plants and nrt1.1 mutants under K⁺-sufficient conditions (Supplemental Fig. S18, A and B). Similarly, in response to low-K⁺ treatment, we found there to be a comparable number of lateral roots in these plant lines. In contrast, however, the average lateral root length of nrt1.1 mutants was clearly less than that of Col-0 plants (Supplemental Fig. S18B). Given that the lack of NRT1.1 had little effect on average lateral root length under our K⁺-sufficient condition, reduction in the average lateral root length in nrt1.1 mutants under low-K⁺ conditions is probably attributable to a reduced influx of K⁺ into root cells (Fig. 4C). Moreover, shorter lateral roots may in turn further reduce K⁺ uptake by the roots of nrt1.1 mutants.

Our observations that both the uptake and root-to-shoot allocation of K⁺ were significantly enhanced by NRT1.1 under K⁺-limiting conditions (Figs. 2B and 4D) indicate that NRT1.1 plays a pivotal role in conferring plant tolerance to K⁺ deficiency (Fig. 1, A, E, and F). Owing to the limited availability of K⁺ in most natural soils, plants often suffer from K⁺-deficiency stress (Wang and Wu, 2013), and consequently, they have evolved complex signaling and physiological regulatory networks that facilitate adaptation to K⁺-deficient environments, which thereby contributes to survival under conditions of K⁺-deficiency stress (Ashley et al., 2006; Tsay et al., 2011). We found that NRT1.1-mediated NO₃⁻ uptake was significantly upregulated in response to low-K⁺ stress (Fig. 3C), and taking into consideration the role of NRT1.1 in improving both the root uptake and root-to-shoot allocation of K⁺ in plants, the upregulated activity of NRT1.1 in response to low-K⁺ stress could be deemed an adaptive mechanism contributing to plant survival in K⁺-deficient environments. Consequently, with respect to crop cultivation, it will be highly desirable to design practical methods aimed at enhancing NRT1.1-mediated NO₃⁻ uptake and increasing the utilization efficiency of K fertilizers. Currently, however, it remains uncertain how low-K⁺ stress promotes upregulation of NRT1.1 activity. In this study, we observed that the rate of root NO₃⁻ uptake in low-K⁺ medium was clearly lower than that in K⁺-sufficient medium (Fig. 3C), and thus it is conceivable that an insufficient K⁺ supply might result in mild nitrogen starvation in plants, which would favor upregulation of NRT1.1 to compensate for the reduced NO₃⁻ uptake. Nevertheless, further studies are required to experimentally verify these assumptions.

In conclusion, in this study, we showed that NO₃⁻ transport activity of NRT1.1 plays an important role in the K⁺ nutrition of plants. Specifically, NRT1.1 expressed in the root epidermis-cortex functions by coordinating with K⁺ uptake channels/transporters, such as AKT1, HAK5, and KUP7, to enhance root uptake of K⁺ from the growth medium via an H⁺-consuming mechanism during NRT1.1-mediated NO₃⁻ uptake, whereas NRT1.1 expressed in the root central vasculature interacts with the channels/transporters that load K⁺ into the xylem, including SKOR and KUP7, to facilitate root-to-shoot allocation of K⁺ (Fig. 8). Although the molecular mechanisms that underlie the interactions between NRT1.1 and K⁺ transporters/channels remain to be elucidated, the findings of this study have enabled us to define the physiological relevance of the interactions among these transporters. In China, current agricultural practices typically entail excessive application of N and K fertilizers, which not only reduces the utilization efficiency of these fertilizers but can also result in environmental pollution (Guo et al., 2010; Zhang, 2017). Our findings indicate that by designing practical methods to enhance the activity of NRT1.1 homologs in different crops, it might be possible to simultaneously improve utilization efficiency of both N and K fertilizers in agricultural production.

Figure 8.

Schematic model depicting how NRT1.1 responds to low-K⁺ stress in Arabidopsis. Low-K⁺ conditions promote upregulated expression of the NRT1.1 gene and encoded protein in the root epidermis-cortex and central vasculature. The H⁺/NO₃⁻ symport activity of NRT1.1 across the plasmalemma of epidermis-cortex cells balances the H⁺-ATPase-mediated H⁺ efflux, which contributes to maintaining a suitable rhizospheric pH for K⁺ uptake channels/transporters, such as AKT1, HAK5, and KUP7, thereby enhancing root K⁺ uptake. The common component CIPK23 kinase, which modulates the uptake activity of AKT1 and HAK5, as well as the affinity of the NO₃⁻ transporter NRT1.1, may play a role in coordinating K⁺ uptake. NRT1.1 expressed in the root central vasculature cells also interacts with channels/transporters that load K⁺ into the xylem, such as SKOR and KUP7, to facilitate the root-to-shoot allocation of K⁺; however, the mechanisms underlying the coordination of this process remain to be clarified.

MATERIALS AND METHODS

Plant Materials

Arabidopsis (Arabidopsis thaliana) mutants chl1-5 (Huang et al., 1996), chl1-9 (Ho et al., 2009), nrt1.1-1 (Mao et al., 2014), nrt1.2 (cs859605), nrt2.1 (cs859604), nrt2.2 (salk_043543), nrt2.5 (GK-213H10.06), akt1 (salk_071803), hak5-3 (salk_130604), kup7 (cs805085), and skor-2 (cs2103489), and the pNRT1.1::NRT1.1-GFP transgenic plants are in an ecotype Columbia (Col-0) background, whereas the chl1-6 (cs6154) and nrt2.4 (cs27332) lines are in a Landsberg erecta (Ler) background. Seeds of the chl1-5 and pNRT1.1::NRT1.1-GFP lines were kindly donated by Dr. Philippe Nacry (Biochimie et Physiologie Moléculaire des Plantes). The double mutants nrt1.1-1/akt1, nrt1.1-1/hak5-3, nrt1.1-1/kup7, and nrt1.1-1/skor-2 were generated by crossing nrt1.1-1 with akt1, hak5-3, kup7, and skor-2, respectively. The mutants were verified using the primers listed in Supplemental Table S1.

Plant Culture

Seeds were surface-sterilized and sown on basal agar medium containing 1% (w/v) Suc and 0.9% (w/v) agar. The complete nutrients in the basal agar medium were composed of 1.125 mm Ca(NO₃)₂, 500 μm CaCl₂, 500 μm MgSO₄, 750 μm NaH₂PO₄, 375 μm (NH₄)₂SO₄, 25 μm Fe-EDTA, 10 μm H₃BO₃, 0.5 μm MnSO₄, 0.5 μm ZnSO₄, 0.1 μm CuSO₄, and 0.1 μm (NH₄)₆Mo₇O₂₄. Conditions in the growth chambers were as follows: a 14 h/10 h light-dark cycle, light intensity of 100 μmol m⁻² s⁻¹, and 75% relative humidity. After germination for 4 d, the seedlings were transferred to basal agar media supplemented with 6, 0.2, or 0 mm NO₃⁻ and 2 or 0.05 mm K⁺, as described in the figure legends. The levels of NO₃⁻ and K⁺ in the media were adjusted using Ca(NO₃)₂ and KCl, respectively. Unless otherwise specified, the initial pH of the growth medium was adjusted to 6.5. For the pH buffer treatments, 0.05% (w/v) MES, 0.02% (w/v) Bis-Tris, or 0.03% (w/v) HEPES was added to the agar medium, and the pH was adjusted to the required value (see Fig. 7 and Supplemental Figs. S12–S14).

Phenotypic Analyses

After 8 d of growth on agar medium containing 2 or 0.05 mm K⁺, plants were photographed and fresh weight and primary root length of plants were recorded. The chlorophyll content of fresh leaves was extracted using 80% (v/v) acetone and measured colorimetrically according to the method described by Choi et al. (2014). In addition, a pulse amplitude-modulating imaging fluorometer (IMAGING-PAM, Walz) was used to analyze chlorophyll fluorescence. Briefly, after a 20-min dark adaption, the leaves were arranged on the fluorometer, and the maximum quantum efficiency (F_v/F_m) and ETR through PSII were monitored.

Measurement of K⁺ Contents

Harvested plants were separated into shoots and roots, which were dried at 75°C for 48 h and then wet-digested as described by He et al. (2017). The digests were diluted with ultrapure water, and the K⁺ contents were analyzed using microwave plasma-atomic emission spectroscopy (Agilent Technologies).

GFP Analysis

NRT1.1-GFP expression in the roots of pNRT1.1::NRT1.1-GFP plants was observed using an Eclipse Ni epifluorescence microscope (excitation at 488 nm; emission at 525–550 nm; Nikon). A camera attached to the microscope was used to image green fluorescence in the roots.

Complementation of nrt1.1-1 with pSultr1;2::NRT1.1 and pPHO1::NRT1.1

The 2-kb genomic fragments upstream of the PHO1 and Sultr1;2 initiation codons were PCR-amplified from Arabidopsis genomic DNA, and the NRT1.1 coding region was amplified from Arabidopsis (Col-0) cDNA using the primer pairs listed in Supplemental Table S1. The PHO1-NRT1.1 and Sultr1;2-NRT1.1 fragments thus generated were cloned into a pCAMBIA1300 vector using EcoRI and SalI restriction sites. The two constructs were used to transform nrt1.1-1 mutant plants via floral-dip infiltration using Agrobacterium tumefaciens strain GV3101, and we subsequently used homozygous T₃ transgenic plants.

Measurement of NO₃⁻ and K⁺, and Rb⁺ uptake

Seedlings were precultured in complete nutrient medium supplemented with 6 mm of NO₃⁻ and 2 or 0.05 mm of K⁺ for 3 d and then transferred to 5-cm-diameter plates to determine the K⁺ and NO₃⁻ fluxes in the meristematic, elongation, and maturation zones of roots using a noninvasive microelectrode ion flux measurement system (SIET IPA-2; Applicable Electronics). Measurements were performed as described by Hawkins et al. (2008) and Li et al. (2012). Briefly, microelectrodes (2- to 4-μm aperture) were pulled from 1.5-mm borosilicate glass capillaries and silanized with N,N-dimethyltrimethylsilylamine. The electrodes were initially backfilled with 100 mm KCl for K⁺ measurement and 500 mm KNO₃ plus 100 mm KCl for NO₃⁻ measurement to a length of ∼1 cm from the tip. Thereafter, the electrode tips were front filled with ∼80 μm columns of K⁺-selective liquid ion exchange cocktails (99373, Sigma-Aldrich), or 25 μm columns of NO₃⁻-selective liquid ion exchange cocktails containing 0.5% (w/v) methyltridodecylammoniumnitrate, 0.084% (w/v) methyltriphenylphosphonium bromide, and 99.416% (w/v) n-phenyloctylether (Plassard et al. 2002). The electrodes were computer-controlled and positioned close to the root surface to detect diffusion potentials outside of the membrane. The testing solution contained 6 mm NO₃⁻ and 2.0 or 0.05 mm K⁺, and the composition of other nutrients was identical to that in the medium used for plant preculture. Ion concentrations were calculated from its electrochemical potential. For Rb⁺ uptake, 4-day-old seedlings were grown on complete nutrient medium containing 2.0 or 0.05 mm K⁺, which was replaced with Rb⁺ for 12 h, after which the plants were harvested and analyzed for Rb⁺ content.

Transcription Analysis

Total RNA in roots was extracted using RNAisoPlus (TaKaRa) and was treated with DNase I to remove contaminant genomic DNA. First-strand complementary DNA (cDNA) was synthesized from genomic DNA-free RNA using a PrimeScript RT reagent kit (TaKaRa), after which the transcript levels of the corresponding genes were measured using qPCR (MJ Research) with TB Green Premix Ex Taq II (TaKaRa). The primers used for amplification are listed in Supplemental Table S1.

RNA Sequencing and Functional Enrichment Analysis

Total RNA was extracted from the roots of Col-0 and nrt1.1-1 plants using MagZol Reagent (Magen) according to the manufacturer’s protocol. Sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), the quality of which was assessed using an Agilent 2100 Bioanalyzer. Sequencing was performed using a Hiseq Xten sequencer (Illumina) at RIBOBIO (Guangzhou, China). Raw sequencing reads were quality controlled and trimmed using Trimmomatic tools and FastQC, and the clean reads thus obtained were aligned to The Arabidopsis Information Resource TAIR10 reference genome using HISAT2. Significantly differentially expressed genes were assessed based on an adjusted P-value threshold of <0.05 and |log₂(fold change)| of >1 using DEGseq (Wang et al., 2010). Functional annotation was conducted based on Gene Ontology (GO) analysis, focusing on all genes associated with K⁺ uptake, translation, and homeostasis (e.g., GO:0006813 K⁺ ion transport). Differences in gene expression patterns were depicted by generating heat maps using the R package ComplexHeatmap (Gu et al., 2016).

Grafting of Arabidopsis Plants

Grafting of nrt1.1-1 mutants and Col-0 plants was performed following the protocol described by Andersen et al. (2014). Briefly, seedlings to be grafted were grown on basal agar medium for 5 d. Having removed the cotyledons, the seedlings were separated into scions and rootstocks using a sterilized blade. Scions were then grafted onto the corresponding rootstocks on fresh agar plates using a stereomicroscope. The grafted seedlings were left undisturbed for a further 5 to 7 d to form a graft union. Adventitious roots emerging at or above the graft union were removed, and the successfully grafted seedlings were subsequently used as described (see Fig. 2).

Determination of pH in the Agar Rooting Medium

The pH of the agar rooting medium was determined as described by Zhu et al. (2019). Measurements were taken from agar in five replicate plates per treatment, each of which contained 15 seedlings. Briefly, the agar rooting medium was collected into a 50 mL centrifuge tube, which was frozen at −20°C overnight and then thawed at room temperature to release the aqueous phase from the agar. The mixture was then filtered at room temperature and the pH of the filtrate was determined using a pH electrode.

Yeast Complementation Assay

The protein-coding sequences of NRT1.1 and AKT1 were PCR amplified from Col-0 cDNA using the primers listed in Supplemental Table S1. NRT1.1 and AKT1 were cloned in the yeast expression vector pDR196 and then transformed into the R5421 strain of yeast (trk1Δ and trk2Δ) using the lithium acetate method (Ito et al., 1983). Thereafter, yeast cells were precultured overnight at 30°C in 2 mL liquid YPDA medium containing 100 mm KCl, and the following day, the cells were collected, washed three times with ultrapure water, re-suspended in ultrapure water to an OD₆₀₀ of 1, and then 10-fold serially diluted from an OD₆₀₀ of 1 to 10⁻³. Aliquots (10 μL) of each dilution were dropped onto Arg phosphate agar plates supplemented with different concentrations of K⁺ and adjusted using KCl. The plates were then incubated at 30°C for 2 d.

Root Growth Analysis

Plant roots were separated from the agar medium and then briefly rinsed three times with distilled water. The root system was carefully spread in a tray of water and scanned with a scanner connected to a computer using WinRhizo (Regent Instruments). Root growth parameters were determined after analysis of the scanned images using Image J software.

Statistical Analyses

Data were analyzed by one-way and two-way ANOVA with Student’s t-test or a least significant difference (LSD) test. P < 0.05 was considered significant.

Accession Numbers

The sequences of genes examined in this study can be found in The Arabidopsis Information Resource data library under accession numbers AT1G12110 (NRT1.1), AT1G69850 (NRT1.2), AT1G08090 (NRT2.1), AT1G08100 (NRT2.2), AT5G60770 (NRT2.4), AT1G12940 (NRT2.5), AT2G26650 (AKT1), AT4G13420 (HAK5), AT5G09400 (KUP7), and AT3G02850 (SKOR).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Root growth of Arabidopsis Col-0, nrt1.1-1, and chl1-5 plants in response to low-K⁺ stress.

• Supplemental Figure S2. Comparisons of the growth and K⁺ contents of Arabidopsis Ler and chl1-6 plants.

• Supplemental Figure S3. Growth phenotypes of Arabidopsis Col-0, chl1-9, and chl1-5 plants subjected to low-K⁺ stress.

• Supplemental Figure S4. Growth responses of Arabidopsis Col-0, nrt1.2, nrt2.1, nrt2.2, nrt2.5, Ler, and nrt2.4 plants to low-K⁺ stress.

• Supplemental Figure S5. Comparisons of the growth and K⁺ contents of Arabidopsis Col-0 and nrt1.1 plants grown on low-nitrate medium.

• Supplemental Figure S6. Comparison of low-K⁺ sensitivity between the chl1-5 mutant and a complementation line (COM; pNRT1.1::NRT1.1-GFP/chl1-5).

• Supplemental Figure S7. Effect of low-K⁺ stress on the expression of NRTs in the roots of Arabidopsis Col-0 plants.

• Supplemental Figure S8. Expression of NRT1.1 in the roots of complementation lines pSultr1;2::NRT1.1 and pPHO1::NRT1.1.

• Supplemental Figure S9. Neither regulation of gene expression nor K⁺ transport activity explains the improvement of K⁺ nutrition in Arabidopsis by NRT1.1.

• Supplemental Figure S10. Comparison of growth among Arabidopsis nrt1.1/akt1, nrt1.1/hak5-3, nrt1.1/kup7, and nrt1.1/skor-2 double mutants and the corresponding single mutants and wild-type plants.

• Supplemental Figure S11. Effect of pH on the K⁺ content of Arabidopsis Col-0 plants and nrt1.1 mutants.

• Supplemental Figure S12. Comparison of growth between Arabidopsis Col-0 and nrt1.1 plants under MES treatment.

• Supplemental Figure S13. pH buffering treatment enhances the Rb⁺ uptake rate of Arabidopsis nrt1.1 mutants.

• Supplemental Figure S14. Treatment with the pH buffer Bis-Tris improves the K⁺ nutrition of Arabidopsis nrt1.1 mutants.

• Supplemental Figure S15. Comparison of the growth and K⁺ content of Arabidopsis Col-0 and nrt1.1 plants in nitrate-free medium.

• Supplemental Figure S16. K⁺ content of Arabidopsis Col-0, nrt1.2, nrt2.1, nrt2.2, nrt2.5, Ler, and nrt2.4 plants.

• Supplemental Figure S17. Roles of K⁺ channels/transporters in NRT1.1-improved K⁺ nutrition.

• Supplemental Figure S18. Changes in the root morphology of Arabidopsis Col-0 plants and nrt1.1 and chl1-5 mutants in response to low-K⁺ stress.

• Supplemental Table S1. Primers used in this study