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Plants have evolved a large array of transporters and channels that are responsible for uptake, source-to-sink distribution, homeostasis and signaling of nitrate (NO3−), which is for most plants the primary nitrogen source and a growth-limiting macronutrient. To optimize NO3− uptake in response to changing NO3− concentrations in the soil, plants are able to modify their root architecture. Potassium is another macronutrient that influences the root architecture. We recently demonstrated that the Arabidopsis NO3− transporter NPF7.3/NRT1.5, which drives root-to-shoot transport of NO3−, is also involved in root-to-shoot translocation of K+ under low NO3− nutrition. Here, we show that K+ shortage, but not limiting NO3− supply, causes in nrt1.5 mutant plants an altered root architecture with conspicuously reduced lateral root density. Since lateral root development is influenced by auxin, we discuss a possible involvement of NPF7.3/NRT1.5 in auxin homeostasis in roots under K+ deprivation.
In most soils, nitrate (NO3−) is the major inorganic nitrogen source for higher plants, and its uptake, translocation, assimilation and remobilization determine plant productivity in terms of plant health and yield formation.1 In Arabidopsis thaliana members of four protein families accomplish NO3− uptake and allocation to individual plant organs.2 The largest of these families is the NPF (nitrate transporter1/peptide transporter - NRT1/PTR) family3 which consists of 53 members in Arabidopsis thaliana. Two NPF proteins were shown to function as bidirectional NO3− influx/efflux transporters involved in root-to-shoot translocation of this important nutrient. NPF6.3/CHL1/NRT1.1 (subsequently termed NRT1.1) is a nitrate transceptor and auxin transporter that controls various physiological and gene regulatory responses to NO3− and represses lateral root development under NO3− deficiency.4,5 The other is the NPF7.3/NRT1.5 protein (subsequently termed NRT1.5) which was characterized as a low-affinity bidirectional NO3− transporter driving xylem loading and root-to-shoot translocation of NO3−.6
We recently found that NRT1.5 is also involved in potassium (K+) homeostasis in shoots by affecting K+ root-to-shoot translocation in a NO3− dependent manner.7 Lately the same phenomenon was reported in a different study.8 Lack of NRT1.5 in roots results in K+ deficiency in shoots under low NO3− nutrition, whereas the whole root elemental composition remains unchanged. Our results indicate that NRT1.5 is an important component linking the NO3− and K+ signaling pathways.
It is well known that the availability and balance of nutrients, particularly nitrate, phosphate and potassium, affects the root system development and architecture by modulating growth rate and morphology of primary roots (PR), lateral roots (LR) and root hairs.4,9-13 For example, low NO3− availability conditionally stimulates10 or represses LR elongation,4,13 whereas K+ deficiency causes shortening of primary root (PR) as well as total root length.14 Potassium is the most abundant and important cation in plants and is critically involved in cell expansion processes like stomatal opening and cell growth in roots.15 The K+ nutritional status is linked to the ethylene, cytokinin, jasmonic acid and auxin signaling pathways, thus leading to developmental and physiological alterations.16-18 Moreover, it presumably influences plant disease resistance.19 When grown under limiting K+ supply, total root size was reduced in 26 investigated natural Arabidopsis accessions, but the degree of reduction and the root architectural response to K+ limitation strongly differed between accessions.20
Drechsler et al. recently discovered that nrt1.5 mutant plants, when growing under low N fertilization conditions, exhibit a pleiotropic shoot phenotype and early leaf senescence symptoms, that are caused by K+ deficiency in the shoot organs.7 However, the NRT1.5 gene is mainly expressed in root pericycle cells which surround the xylem.6 This prompted us to investigate root development of nrt1.5 mutants under varying NO3− and K+ supply. Arabidopsis Col-0 and nrt1.5 seeds were germinated for 5 days on 0.5 × MS medium (containing ~30 mM N) and then transferred to modified 0.5 × MS medium with reduced N and K+ concentrations. On medium containing 1 mM K+, reducing the NO3− concentration from 2 mM to 0 mM resulted in wild-type as well as nrt1.5 seedlings in a dramatic impairment of shoot development, decreased chlorophyll biosynthesis and accumulation of anthocyanins (Fig. 1A).7 Shoot and root fresh weight and root architecture of wild-type and nrt1.5 mutants are virtually identical though (Fig. 1A–C). Similarly, on medium containing 2 mM K+ and 1 mM NO3−, mutant and wild-type seedlings are phenotypically indistinguishable and show no morphological abnormalities in shoots or roots (Fig. 2A).
Remarkably, in Col-0 wild-type plants the absence of NO3− does not affect LR density (Fig. 1C). This contrasts with the phenotype of seedlings that were sown and germinated on medium lacking NO3−. Under these conditions LR density is significantly reduced, although the LR primordia density is not altered.4,13 At low NO3− supply NRT1.1 prevents auxin accumulation in LR primordia, thereby repressing development of LRs.4 In our experiment, after 5 days germination on 0.5 × MS medium the LRs have emerged already before the plants are transferred to NO3− free medium, suggesting that under NO3− deficiency NRT1.1 suppresses the outgrowth of LR primordia but not or only marginally the elongation of emerged LRs. This conclusion is consistent with the findings of Linkohr and colleagues that the LR elongation response to varying NO3− concentrations is not affected in auxin-resistant mutants and thus wild-type auxin signaling is not required for mediating LR elongation.10
Other than NO3− deficiency, lowering the K+ concentration in the medium to less than 1 mM perturbs development of the nrt1.5 mutants more severely than wild-type plants. The mutant's roots and shoots gain only 50% and 60% fresh weight relative to wild-type, respectively (Fig. 2B). Notably, the root architecture of the mutants is conspicuously altered under K+ deficiency. The primary roots are slightly, but significantly shorter and the lateral root density is reduced to approximately 60% (Fig. 2A,C). We have previously shown that expression of NRT1.5 by the PHO1 promoter, which has a comparable root specific activity pattern as the NRT1.5 promoter but was more active in the transgenic plants, successfully complements the K+ deficiency phenotype in shoots of nrt1.5-5 plants.7 We therefore examined outgrowth and development of LRs in two independent PHO1p:NRT1.5 complementation lines on medium with 0 mM K+/1.0 mM NO3− supply. Indeed, after seven days the LR density of both PHO1p:NRT1.5 lines is identical to wild-type plants (Fig. 3). These results demonstrate that under K+ limiting conditions, but not under NO3− deficiency, the nitrate transporter NRT1.5 is required for normal root development.
Lin and colleagues reported that NRT1.5 is predominantly expressed in root pericycle cells adjacent to the protoxylem but they did not investigate LR development in detail.6 Considering the reduced LR density in nrt1.5 mutants under K+ deficiency, we reexamined the activity and tissue specificity of the NRT1.5 promoter in primary and lateral roots. In ten independent Arabidopsis Col-0 transformants carrying the ß-glucuronidase (uidA) gene under the control of a 2.4 kb NRT1.5 promoter fragment, we observed largely identical GUS staining patterns. In 15 days old seedlings, NRT1.5 promoter activity is detected throughout the PR vascular cylinder, while the root cortex is not stained (Fig. 4A). GUS staining is most intense at lateral root primordia (Fig. 4A–B), whereas in the newly developing lateral roots at first the vascular tissue, root cap, elongation zone and meristematic zone are not stained (Fig. 4B, black arrowheads). With increasing age, NRT1.5 promoter activity spreads from the lateral root primordia also along the lateral root vasculature (Fig. 3B, white arrowhead). These results suggest that transport processes regulated or performed by NRT1.5 are important determinants for lateral root outgrowth.
In Arabidopsis the potassium transporter HAK5 and the shaker-family potassium channel AKT1 are the two principal K+ uptake systems at moderate to low K+ supply.21 At very low external K+ concentrations (<10 µM) the high-affinity K+ transporter HAK5 is the only protein capable of taking up K+.22 By K+ deprivation HAK5, but not AKT1, is transcriptionally upregulated in Col-0 roots.23,24 Drechsler et al. found that HAK5 is approximately 3-fold downregulated in roots of nrt1.5 mutant plants.7 It is therefore conceivable that at limiting external K+ supply the reduced expression of HAK5 in nrt1.5 roots leads to K+ shortage in the roots. This shortage could indirectly be responsible for the decreased LR density phenotype of nrt1.5 plants at low K+ supply by repressing the K-dependent expression of the auxin signaling-modulating transcription factor MYB77.25 Indeed, the phenotype of myb77 mutants resembles the nrt1.5 phenotype at low K+ supply.26 Auxin is known to control LR development through multiple auxin-signaling components27 and it is therefore tempting to speculate that NRT1.5 could be involved in K+-dependent hormone homeostasis. NRT1.1 is known to control LR primordia outgrowth by preventing auxin accumulation in LR primordia at low NO3− supply. In roots of adult nrt1.5-5 mutant plants hydroponically grown under low NO3− supply NRT1.1 expression is not altered.7 To investigate whether the knock-out of NRT1.5 affects NRT1.1 expression under K+ limitation, we determined NRT1.1 expression in roots of the Col-0 and nrt1.5-5 plants shown in Fig. 2A by qRT-PCR. However, NRT1.1 expression is not significantly altered in nrt1.5-5 relative to wild-type plants in 0 mM K+/1.0 mM NO3− or 2.0 mM K+/1.0 mM NO3−, respectively (data not shown). It is therefore unlikely that the decreased LR density phenotype of nrt1.5 plants at low K+ supply is caused indirectly via modulation of auxin distribution by NRT1.1. Whether NRT1.5 is directly or indirectly involved in auxin transport remains to be investigated in future studies.
No potential conflicts of interest were disclosed.
We thank Nikolaus von Wirén and Alexis Kasaras for helpful comments on the manuscript.
This research was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR948, KU715/10-2) and fellowships of the China Scholarship Council to Y.Z. and the Studienstiftung des deutschen Volkes to N.D.