Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner

Root development is strongly affected by the plant's nutritional status and the external availability of nutrients. Employing split-root systems, we show here that local ammonium supply to Arabidopsis thaliana plants increases lateral root initiation and higher-order lateral root branching, whereas the elongation of lateral roots is stimulated mainly by nitrate. Ammonium-stimulated lateral root number or density decreased after ammonium or Gln supply to a separate root fraction and did not correlate with cumulative uptake of (15)N-labeled ammonium, suggesting that lateral root branching was not purely due to a nutritional effect but most likely is a response to a sensing event. Ammonium-induced lateral root branching was almost absent in a quadruple AMMONIUM TRANSPORTER (qko, the amt1;1 amt1;2 amt1;3 amt2;1 mutant) insertion line and significantly lower in the amt1;3-1 mutant than in the wild type. Reconstitution of AMT1;3 expression in the amt1;3-1 or in the qko background restored higher-order lateral root development. By contrast, AMT1;1, which shares similar transport properties with AMT1;3, did not confer significant higher-order lateral root proliferation. These results show that ammonium is complementary to nitrate in shaping lateral root development and that stimulation of lateral root branching by ammonium occurs in an AMT1;3-dependent manner.     

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.     

Functional analysis of an Arabidopsis T-DNA "knockout" of the high-affinity NH4(+) transporter AtAMT1;1

NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.     

Regulation of NH4+ transport by essential cross talk between AMT monomers through the carboxyl tails

Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH4+ acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH4+ transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH4+ transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH4+ transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium.     

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in planta. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb⁺ uptake experiments reveal a reduced uptake in csi52 in both the high- and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wild-type and mutant. Rb⁺ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs⁺ than the low-affinity pathway with K_1/2 values for Cs⁺ of around 1.3 and 0.2 mM, respectively. Furthermore, K⁺ starvation leads to a larger relative increase in high-affinity K⁺ uptake in the mutant than the wild-type. The results demonstrate the Cs⁺ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs⁺ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K⁺-starvation conditions will endow the mutant with a higher degree of overall Cs⁺ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K⁺ uptake.     

AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots

Despite the fact that urea is a ubiquitous nitrogen source in soils and the most widespread form of nitrogen fertilizer used in agricultural plant production, membrane transporters that might contribute to the uptake of urea in plant roots have so far been characterized only in heterologous systems. Two T-DNA insertion lines, atdur3-1 and atdur3-3, that showed impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transporter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing AtDUR3-promoter:GFP constructs, promoter activity was upregulated under nitrogen deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. Protein gel blot analysis of two-phase partitioned root membrane fractions and whole-mount immunolocalization in root hairs revealed the plasma membrane to be enriched in AtDUR3 protein. Expression of the AtDUR3 gene in nitrogen-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher accumulation of urea in roots of wild-type plants relative to atdur3-1 and atdur3-3 confirmed that urea was the substrate transported by AtDUR3. Influx of 15N-labeled urea in atdur3-1 and atdur3-3 showed a linear concentration dependency up to 200 microM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 microM. The results indicate that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggest that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.     

High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.     

Uniport of NH4+ by the root hair plasma membrane ammonium transporter LeAMT1;1

The transport of ammonium/ammonia is a key process for the acquisition and metabolism of nitrogen. Ammonium transport is mediated by the AMT/MEP/Rh family of membrane proteins which are found in microorganisms, plants, and animals, including the Rhesus blood group antigens in humans. Although ammonium transporters from all kingdoms have been functionally expressed and partially characterized, the transport mechanism, as well as the identity of the true substrate (NH(4+) or NH(3)) remains unclear. Here we describe the functional expression and characterization of LeAMT1;1, a root hair ammonium transporter from tomato (Lycopersicon esculentum) in Xenopus oocytes. Micromolar concentrations of external ammonium were found to induce concentration- and voltage-dependent inward currents in oocytes injected with LeAMT1;1 cRNA, but not in water-injected control oocytes. The NH(4+)-induced currents were more than 3-fold larger than methylammonium currents and were not subject to inhibition by Na(+) or K(+). The voltage dependence of the affinity of LeAMT1;1 toward its substrate strongly suggests that charged NH(4+), rather than NH(3), is the true transport substrate. Furthermore, ammonium transport was independent of the external proton concentration between pH 5.5 and pH 8.5. LeAMT1;1 is concluded to mediate potential-driven NH(4+) uptake and retrieval depending on root membrane potential and NH(4+) concentration gradient.