A novel morphological response of maize (Zea mays) adult roots to heterogeneous nitrate supply revealed by a split‐root experiment

Approximately 35–55% of total nitrogen (N) in maize plants is taken up by the root at the reproductive stage. Little is known about how the root of an adult plant responds to heterogeneous nutrient supply. In this study, root morphological and physiological adaptations to nitrate‐rich and nitrate‐poor patches and corresponding gene expression of ZmNrt2.1 and ZmNrt2.2 of maize seedlings and adult plants were characterized. Local high nitrate (LoHN) supply increased both lateral root length (LRL) and density of the treated nodal roots of adult maize plants, but only increased LRL of the treated primary roots of seedlings. LoHN also increased plant total N acquisition but not N influx rate of the treated roots, when expressed as per unit of root length. Furthermore, LoHN markedly increased specific root length (m g^?1) of the treated roots but significantly inhibited the growth of the lateral roots outside of the nitrate‐rich patches, suggesting a systemic carbon saving strategy within a whole root system. Surprisingly, local low nitrate (LoLN) supply stimulated nodal root growth of adult plants although LoLN inhibited growth of primary roots of seedlings. LoLN inhibited the N influx rate of the treated roots and did not change plant total N content. The gene expression of ZmNrt2.1 and ZmNrt2.2 of the treated roots of seedlings and adult plants was inhibited by LoHN but enhanced by LoLN. In conclusion, maize adult roots responded to nitrate‐rich and nitrate‐poor patches by adaptive morphological alterations and displayed carbon saving strategies in response to heterogeneous nitrate supply.     

Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms

An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.     

Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges

Plants take up both nitrate and ammonium as main nitrogen (N) sources. Although ammonium is the predominant form in anaerobic-flooded paddy soil, it has been proposed that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere. A two-component system for nitrate transport including NRT2s with a partner protein (NAR2 or NRT3.1) has been identified in Arabidopsis. We report the physiological function of another member of the NAR2 family, OsNAR2.1 in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). OsNAR2.1 was mainly expressed in roots and induced by nitrate and suppressed by ammonium and some amino acids. Knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in the osnar2.1mutants. Both high- and low-affinity nitrate transports were greatly impaired by OsNAR2.1 knockdown. Yeast two hybridization showed that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2, but also with OsNRT2.3a. Taken together, the data demonstrate that OsNAR2.1 plays a key role in enabling the plant to cope with variable nitrate supply.     

Multiple roles of nitrate transport accessory protein NAR2 in plants

Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.Key words: NAR2, NRT2, nitrate transporter, root     

Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.     

Cloning,functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k)

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.     

The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.     

Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina

A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO^– ₃, but not by NH⁺ ₄. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1.     

A two-component high-affinity nitrate uptake system in barley

The analysis of genome databases for many different plants has identified a group of genes that are related to one part of a two-component nitrate transport system found in algae. Earlier work using mutants and heterologous expression has shown that a high-affinity nitrate transport system from the unicellular green algae, Chlamydomonas reinhardtii required two gene products for function. One gene encoded a typical carrier-type structure with 12 putative trans-membrane (TM) domains and the other gene, nar2 encoded a much smaller protein that had only one TM domain. As both gene families occur in plants we investigated whether this transport model has more general relevance among plants. The screening for nitrate transporter activity was greatly helped by a novel assay using (15)N-enriched nitrate uptake into Xenopus oocytes expressing the proteins. This assay enables many oocytes to be rapidly screened for nitrate transport activity. The functional activity of a barley nitrate transporter, HvNRT2.1, in oocytes required co-injection of a second mRNA. Although three very closely related nar2-like genes were cloned from barley, only one of these was able to give functional nitrate transport when co-injected into oocytes. The nitrate transport performed by this two-gene system was inhibited at more acidic external pH and by acidification of the cytoplasm. This specific requirement for two-gene products to give nitrate transport function has important implications for attempts to genetically manipulate this fundamental process in plants.     

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO₃). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation. Received: 4 May 1998 / Accepted: 26 August 1998     

Identification and molecular characterization of Medicago truncatula NRT2 and NAR2 families

Nitrate transporters received little attention to legumes probably because these species are able to adapt to N starvation by developing biological N₂ fixation. Still it is important to study nitrate transport systems in legumes because nitrate intervenes as a signal in regulation of nodulation probably through nitrate transporters. The aim of this work is to achieve a molecular characterization of nitrate transporter 2 (NRT2) and NAR2 (NRT3) families to allow further work that would unravel their involvement in nitrate transport and signaling. Browsing the latest version of the Medicago truncatula genome annotation (v4 version) revealed three putative NRT2 members that we have named MtNRT2.1 (Medtr4g057890.1), MtNRT2.2 (Medtr4g057865.1) and MtNRT2.3 (Medtr8g069775.1) and two putative NAR2 members we named MtNAR2.1 (Medtr4g104730.1) and MtNAR2.2 (Medtr4g104700.1). The regulation and the spatial expression profiles of MtNRT2.1, the coincidence of its expression with that of MtNAR2.1 and MtNAR2.2 and the size of the encoded protein with 12 transmembrane (TM) spanning regions strongly support the idea that MtNRT2.1 is a nitrate transporter with a major contribution to the high‐affinity transport system (HATS), while a very low level of expression characterized MtNRT2.2. Unlike MtNRT2.1, MtNRT2.3 showed a lower level of expression in the root system but was expressed in the shoots and in the nodules thus suggesting an involvement of the encoded protein in nitrate transport inside the plant and/or in nitrate signaling pathways controlling post‐inoculation processes that govern nodule functioning.