Overexpressing NRT2.7 induces nitrate export from the vacuole and increases growth of Arabidopsis

Overexpressing NRT2.7 induces nitrate export from the vacuole and increases growth of Arabidopsis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Overexpressing NRT2.7 induces nitrate export from the vacuole and increases growth of Arabidopsis View ORCID Profile Patrick Armengaud , View ORCID Profile Alexis De Angeli , View ORCID Profile Patrick Berquin , View ORCID Profile Virginie Bréhaut , View ORCID Profile Mickaël Durand , Françoise Daniel-Vedele , View ORCID Profile Anne Krapp , View ORCID Profile Sophie Filleur doi: https://doi.org/10.1101/2024.02.26.582130 Patrick Armengaud 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Armengaud Alexis De Angeli 2 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC) , 91198, Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexis De Angeli Patrick Berquin 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Berquin Virginie Bréhaut 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Virginie Bréhaut Mickaël Durand 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mickaël Durand Françoise Daniel-Vedele 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anne Krapp 1 Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) , 78000, Versailles, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anne Krapp For correspondence: anne.krapp{at}inrae.fr sophie.filleur{at}i2bc.paris-saclay.fr Sophie Filleur 2 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC) , 91198, Gif-sur-Yvette, France 3 Université Paris Cité, UFR Sciences du Vivant , 35 rue Hélène Brion, F-75205 Paris Cedex 13, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sophie Filleur For correspondence: anne.krapp{at}inrae.fr sophie.filleur{at}i2bc.paris-saclay.fr Abstract Full Text Info/History Metrics Preview PDF Abstract Nitrogen nutrition is essential for crop yield but applying fertilizers has detrimental effects on the environment. Compartmenting nitrate into vacuoles is one of the options to develop Nitrogen-efficient crop adapted to less fertilizers. Only few proteins involved in nitrate transport on the tonoplast have been identified. CLCa is the major transporter involved in nitrate storage in Arabidopsis but it can also facilitate nitrate remobilization from the vacuole in guard cells. Several other nitrate transporters amongst NRT2.7 have been localized in this membrane. The transport mechanism of NRT2.7 has not yet been defined as this protein is present mainly in seed cells that are not easily amenable for electrophysiology analysis. Here, we investigated NRT2.7 function through its ectopic overexpression in a clca knock-out mutant. Although the growth diminution of clca on nitrogen sufficient medium was complemented, nitrate homeostasis was not restored by NRT2.7 activity. Moreover, NRT2.7 ectopic overexpression in wild-type background (WT) increased growth under limiting nitrogen supply, suggesting that NRT2.7 stimulates nitrate efflux from vacuoles. This hypothesis was demonstrated by electrophysiological nitrate flux measurements on isolated vacuoles. This discovery of NRT2.7 function and more largely the coupling of vacuolar nitrate fluxes with growth under low nitrate supply, will enable new strategies for engineering better NUE for a more sustainable agriculture. Highlight The overexpression of the nitrate transporter NRT2.7 stimulates growth by increasing the export of nitrate from the vacuole, the main cell compartment for nitrate storage. Introduction Nitrogen (N) is an essential macronutrient acquired by plants from the soil or the air to synthesize amino acids, nucleotides and proteins. Most cultivated plant species rely on soil soluble ammonium and nitrate, the latter being the most abundant oxidized form of nitrogen found in temperate agricultural soils in amounts ranging from 0.1 to 10 mM ( Miller et al ., 2007 ). The effect of N supply on plant growth has long been evidenced and this nutritional benefit was the basis of the fertilizer-driven yield increases. Crops are therefore abundantly provided with nitrogen fertilizers to insure best yield. This practice is at high economic and ecological costs since it has been shown that plants use only 25 to 50 % of nitrogen applied in the field ( Cassman et al ., 2002 ). Because of both nitrate mobility and soil structure, nitrate leaks from the field, contaminates water effluents and stimulates growth of algae and microorganisms leading to ecological problems such as river eutrophication ( Howarth and Marino, 2006 ). To avoid these problems and move toward a more sustainable agriculture, one means is to reduce fertilizer inputs without affecting crop yields by improving plant nitrogen use efficiency (NUE). This objective is challenging since NUE is a complex trait that includes uptake, utilization and remobilization efficiencies of the whole plant or of specific organs such as grains ( Liu et al ., 2022 ). Therefore, research on NUE improvement has been tackled through approaches engineering known molecular players (transporters, enzymes) or heuristically by Quantitative Trait Loci (QTL), Genome-Wide Association Study (GWAS) and transcriptome candidate genes approaches. However, to date, crop varieties with optimized NUE are still rare ( Liu et al ., 2022 ; Melino et al ., 2022 ; Govindasamy et al ., 2023 ). The large number of nitrate transport mechanisms and their multiple level of regulation form a complex network coordinating nitrate fate and homeostasis. The latter is illustrated by remarkably stable cytosolic nitrate levels ranging between 3 and 5 mM ( Cookson et al ., 2005 ). Once absorbed into root cells, nitrate has three possible fates ( Dechorgnat et al ., 2011 ). A first one is a direct export to vascular tissue partially through xylem associated nitrate transporters. A second one is its assimilation where cytosolic nitrate is reduced first by the nitrate reductase to nitrite and then by the plastidial nitrite reductase to form ammonium. Ammonium is then incorporated into organic acids to produce amino acids. The third fate of cytosolic nitrate is vacuolar storage. The vacuolar compartment plays important roles in controlling of the plant nitrate status as vacuolar nitrate accumulation has two major functions ( Han et al ., 2016 ; Hodin et al ., 2023 ). First, vacuolar nitrate represents a temporary reserve of N allowing a near constant cytoplasmic provision independently of external fluctuations as nitrate concentrations in this compartment can reach up to 80 mM ( van der Leij et al ., 1998 ; Cookson et al ., 2005 ). Second, vacuolar nitrate acts as an osmoticum contributing to the osmotic potential, including during stomata movements ( McIntyre, 1997 ; Wege et al ., 2014 ; Hodin et al ., 2023 ). The first nitrate/proton exchanger mediating nitrate influx into the vacuole, CLCa, was characterized in Arabidopsis ( De Angeli et al ., 2006 ). This protein is part of a family of seven members in this species. CLCa and CLCb were shown to preferentially exchange nitrate over chloride against proton ( De Angeli et al ., 2006 ; Bergsdorf et al ., 2009 ; von der Fecht-Bartenbach et al ., 2010 ; Wege et al ., 2010 ; Barbier-Brygoo et al ., 2011 ). Both of these exchangers are located on the vacuolar membrane. CLCa activity accounts for up to 50% of vacuolar nitrate content but can also be involved in nitrate efflux from the vacuole in stomata depending of its state of phosphorylation ( Geelen et al ., 2000 ; Wege et al ., 2014 ). Impaired nitrate flux into vacuoles through CLCa was shown to reduce nitrate uptake, nitrate content and expression level of several nitrate transporter genes but to increase nitrate assimilation and consequently NUE ( Monachello et al ., 2009 ; Hodin et al ., 2023 ). As for CLCb, its participation in nitrate accumulation could not been demonstrated as nitrate contents are at the same level in wild type and clcb KO mutant when plants are grown in sufficient nitrate-condition ( von der Fecht-Bartenbach et al ., 2010 ). However, it was proposed recently that CLCb mediates nitrate efflux from the vacuoles when plants are grown on low nitrate concentrations ( Shi et al ., 2023 ). Next to CLC transporters, some members of NRT2 and NPF transporters family are localized in the vacuolar membrane ( Hu et al ., 2016 ; He et al ., 2017 ; Wang et al ., 2018 ; Lu et al ., 2022 ). Among them, NRT2.7 belongs to the seven members of the NRT2 gene family in Arabidopsis that encode major high affinity nitrate transporters. All NRT2 proteins except NRT2.7 are localized at the plasma membrane. NRT2.7 forms indeed a distinct monophyletic group within the Arabidopsis NRT2 family. The expression of the NRT2.7 gene occurs mainly in seeds, but its expression has been also described for leaves and roots ( Orsel et al ., 2002 ; Okamoto et al ., 2003 ; Chopin et al ., 2007 ). NRT2.7 is able to mediate nitrate fluxes in Xenopus oocytes. The main phenotype of nrt2 .7 knock-out mutants is a greatly decreased nitrate content in seeds which highlights the major roles of NRT2.7 in nitrate accumulation at reproductive stage ( Chopin et al ., 2007 ). Regarding nitrate as a signal, a nutrient or an electro-osmotic charge, further work in characterizing transporters and channels involved in vacuolar fluxes will shed light on the putative sensing and signaling properties of the largest compartment of plant cells. The mechanism of nitrate transport by NRT2.7 was not investigated in planta as this protein is essentially present in seeds. So, in this report, we first investigated the impact of a NRT2.7 overexpression in Arabidopsis on nitrate homeostasis, activities of plasma membrane nitrate uptake systems, growth and vacuolar nitrate currents. Functional characterization of nitrate transporters in planta often requires the genetic removal of the quantitatively most important components to uncover more subtle or fine-tuning mechanisms. For example, the nitrate transport activity of NRT2 family members are masked by the NRT2.1 activity and has to be assessed in the nrt2.1 deficient background ( Chopin et al ., 2007 ; Kiba et al ., 2012 ; Lezhneva et al ., 2014 ). Here we applied a similar strategy and used the clca mutant background in addition to a wild-type background, to test the physiological impact of NTR2.7 activities in planta and to determine by electrophysiology the effect the overexpression of NRT2.7 in clca on vacuolar nitrate currents. We thus elucidate the function of NRT2.7 by asking the following questions: (i) whether or not overexpression of NRT2.7 nitrate transporter would give similar effects as overexpression of the CLCa nitrate exchanger in the clca knock-out genetic background, (ii) how the NRT2.7 overexpression in wild-type background would affect nitrate homeostasis and growth under nitrate sufficient and limiting conditions and, (iii) what are the consequences of NRT2.7 overexpression on nitrate fluxes through the vacuolar membrane. Material and methods Plant material and growth conditions All Arabidopsis thaliana genotypes used in this study were obtained or generated in the Wassilewskija (WS) background. The clca-2 T-DNA knock-out plant line originates from the Versailles collection (FST 171A06, line EAE89] whereas clca-1 was isolated in Geelen et al . (2000) . The CLCa-OX ( clca-2 ) line ( clca-2 knock-out mutant overexpressing CLCa under the 35S promoter of the Cauliflower Mosaic Virus, CaMV) originates from Wege et al . (2010) . The NRT2.7-OX ( clca ) lines ( clca knock-out mutant overexpressing NRT2.7 under the 35S promoter of the CaMV) was obtained by crossing NRT2.7 overexpressors (in the WS background, Chopin et al ., 2007 ) with the clca mutants ( clca-1 and clca-2 ). The vectors used to over-express CLCa and NRT2.7 contain the three regions necessary for the activity of the 35S promoter ( Ow et al ., 1987 ). Seeds were surface-sterilized, stored for 3 days at 4°C to synchronize germination and sown in 120 x 120 mm square Petri dishes containing either 35 or 70 mL nutrient medium to monitor root architecture (4-5 plants/plate) or to produce material for analysis (12-15 plants/plate). Media differed in their nitrate content (0.2|2|20 mM) and consisted respectively of 0.2|0.7|7 mM KNO 3 ; 0|0.65|6.5 mM Ca(NO 3 ) 2 ; 0.5|0.5|1.5 mM MgSO 4 ; 0.625|0.625|0 mM KH 2 PO 4 ; 0|0|1.875 mM NaH 2 PO 4 ; 0.75|1.25|1.25 mM NaCl; 0.65|0.25|0 mM CaCl 2 and 1.05|0.55|0 mM KCl. For all media, micronutrients (in µM) consisted of 42.5 FeNaEDTA, 0.16 CuSO 4 , 45 H 3 BO 3 , 0.015 (NH 4 ) 6 Mo 7 O 24 , 0.01 CoCl 2 , 0.38 ZnSO 4 , 1.8 MnSO 4 . All media were supplemented with 1% sucrose, pH balanced at 5.8 before autoclaving and solidified using 1% agar (Sigma A1296). Petri dishes were sealed using 3M adhesive tape and placed below tubes delivering 65 µE irradiance for 16h/day in a tissue culture room with a thermoperiod day/night of 22/18°C. At 12 days after germination (14 days after sowing), plants were harvested (or sampled for 15 N uptake) between 10 and 12 am (after 3 to 5h light exposure). Growth measurements Root architecture was measured at 3, 6 and 9 days after germination by scanning Petri dishes using a flat-bed scanner (Epson Perfection 4490 Photo) at a 200 dots per inch resolution. Total root length was acquired by analyzing root morphology with the EZ-Rhizo software ( Armengaud et al ., 2009 ). Plant biomass was measured directly on a microbalance (Mettler Toledo XS3DU) for fresh weight. RNA extraction and RT-qPCR analysis RNA was extracted from shoots using the Trizol reagent (Invitrogen) according to the provider recommendations. One microgram was treated with DNase and reverse transcribed using the Quantitect kit (Qiagen) following the manufacturer instructions. For qPCR analysis, specific primers were used for NRT2.7 (5‘-CCTTCATCCTCGTCCGTTTC-3’ (forward) and 5’-AATTCGGCTATGGTGGAGTA-3’ (reverse)) and EF1α ((At5g60390) 5’-CTGGAGGTTTTGAGGCTGGTAT-3’ (forward) and 5’-CCAAGGGTGAAAGCAAGAAGA-3’ (reverse)). The reactions were performed in a Realplex mastercycler (Eppendorf) using commercial SYBR green mastermix (Roche). Samples were subjected to 2 min pre-incubation at 95°C followed by 40 amplification cycles of 15 s at 95°C, 15 s at the melting temperature of the primers set and 20 s at 68°C. NRT2.7 transcript levels were quantified using linear regression of quantified amplicon range and expressed relatively to EF1α . Analysis of nitrate, total carbon, and total N contents Frozen plant material was ground in liquid nitrogen using a Retsch mill grinder (MM301, Retsch GmbH, Germany). For nitrate content determination, an aliquot of 20 mg of frozen powder was used to prepare an ethanol-water extract as described in Cross et al . (2006) . Nitrate content was assayed after its reduction by vanadium(III) combined with detection by the acidic Griess reaction as described in Miranda et al . (2001) . Total carbon (C) and N content were determined using an elemental analyzer (EA, Flash 2000, Thermo Scientific) on aliquots of 0.5 to 2 mg dry weight in tin capsules. EA settings consisted of 20 sec of oxygen flux at 80 mL/min and helium at 140 mL/min for 5 min. 15N nitrate uptake measurements Influx of 15 NO 3 - was assayed as previously described ( Orsel et al ., 2004 ). Two to three 12-day-old plants were first transferred to 0.1 mM CaSO 4 for 1 min. To measure high affinity 15 N nitrate uptake, plants were then transferred to a liquid 0.2 mM nitrate containing medium (the same as described earlier but without sucrose) enriched at 99% with 15 NO 3 - (Sigma). To measure low affinity transport capacity, plants were instead transferred to a 6 mM nitrate medium consisting of 3 mM K 15 NO 3 ; 1.5 mM Ca( 15 NO 3 ) 2 ; 0.5 mM MgSO 4 ; 0.625 mM KH 2 PO 4 ; 1.5 mM NaCl and the same micronutrients as described before. 15 N nitrate uptake was performed for 5 min and plants were finally incubated for 1 min in 0.1 mM CaSO 4 . Roots were then placed into tin capsules, dried at least 48h at 80°C and sent for 15 N elemental analysis using an ANCA–MS system (PDZ Europa, Crewe, UK) at INRAE Montpellier. Nitrate uptake was calculated from the 15 N content of the roots (0.5 to 1.5 mg DW). Each uptake experiment included at least 5 measurements per condition and genotype. Patch-clamp analyses Patch-clamp experiments were made in the whole-vacuole configuration with an access resistance between 1.5-3 MΩ. Patch-clamp recordings were made with an EPC8 amplifier (HEKA, Lambrecht-Pfalz Germany). Data were acquired with an AD/DA converter Instrutech LIH 8+8 under the control of the software PatchMaster (HEKA, Lambrecht-Pfalz Germany) and analysed with FitMaster software (HEKA, Lambrecht-Pfalz Germany). Applied membrane potentials were always corrected by the liquid junction potential ( Neher, 1992 ). Current to voltage characteristics were obtained measuring the amplitude of the stationary current. Measurements were done after waiting that the vacuolar lumen solution was equilibrated with the pipette solution, typically 10 minutes after the establishment of the whole-vacuole configuration. Cytosolic side solutions were changed using a gravity driven perfusion system. Bis-Tris-propane (BTP) was chosen as a large impermeable cation in order to minimize cation currents. The solution used for patch-clamp experiments were (in mM): Nitrate vacuolar solution: 100 BTP, 200 HNO 3 , 0.1 CaCl 2 , 5 MgCl 2 , 5 MES, pH = 5.5. Chloride vacuole solution : 475 MES, 15 HCl, 60.5 BTP, 0.1 CaCl 2 , pH = 5.5. Chloride cytosolic solutions: 7.5 BTP, 15 HCl, 0.1 CaCl 2 , 2 MgCl 2 , 15 MES, pH = 7. Nitrate cytosolic solution: 0.1 Ca(NO 3 ) 2 , 2 Mg(NO 3 ) 2 , 15 MES, pH = 7 with BTP. Osmotic pressures of the cytosolic and vacuolar solutions were adjusted with sorbitol to 640 and 650 mOsm, respectively. Statistical analysis Statistical analyses were conducted using the pairwisePermutationTest function from the rcompanion package in the R environment, except for the electrophysiology results. For those, a Mann-Whitney test was applied using GraphPad Prism 8 software. All the raw data and the results of the statistical analysis can be found in the supplementary table. Results Both CLCa and NRT2.7 overexpression restore growth of the clca-2 mutant To set a suitable genetic background for NRT2.7 functional studies, we characterized the phenotypes of clca mutants on a range of nitrate supplied as a sole nitrogen source in vertical Petri dishes. We used clca-1 and clca-2 knock-outs ( Geelen et al ., 2000 ; De Angeli et al ., 2006 ) which both showed a significant decrease in shoot fresh weight (-13 to -45%) and root growth rate (-8 to -40%) when compared to the WS wild type ( Fig. 1 , Supplementary Fig. S1A, B and S2A, B ). This decrease was more pronounced under non-limiting nitrate supply (2 and 20 mM). As expected, clca mutants showed reduced nitrate content compared to WS under all N supplies in both shoot and root (Supplemental Fig. S1C, D and S2C, D). Download figure Open in new tab Supplementary Fig. S1. clca-2 knock-out mutant phenotype. Wild-type (WS) and clca-2 p lants were grown on vertical Petri dishes for 12 days on 0.2, 2 or 20 mM external nitrate as described in the ‘Materials and Methods’. Shoot fresh weight (A), total root length (B), shoot and root nitrate contents (C and D) were measured in WS (white boxes) and clca-2 (black boxes) after 12 days of growth (A). Data are means ± SEM of 4-5 (A) or 16-20 measurements (B). For nitrate measurements, data are means ± SEM of three technical replicates of a representative experiment. Each point is a pool of at least 50 plants. Different letters at the top of the bars represent significant difference (p value<0.05). Download figure Open in new tab Supplementary Fig. S2. Growth and nitrate content of NRT2.7 overexpressors in the c lca-1 knock-out mutant backgound. Wild type (WS), clca-1 and two over-expressing lines, NRT2.7-Ox1 ( clca-1 ) and NRT2.7-Ox2 ( clca-1 ) were grown on vertical Petri dishes for 12 days on 0.2, 2 or 20 mM external nitrate as described in the ‘Materials and Methods’. Shoot fresh weight was measured after 12 days of growth (A), and total root length were analyzed on 9 day-old plant (B). Data are averages of 4-5 (A) or 11-20 measurements (B). Shoot (C) and root (D) nitrate content in 12 day-old plants. Data are averages of three technical nitrate measurements of a representative experiment. Each point is a pool of at least 50 plants. Vertical bars represent means ± SEM. Different letters at the top of the bars represent significant difference (p value<0.05). Download figure Open in new tab Fig. 1. Growth effect of NRT2.7 overexpression in clca-2 and wild-type backgrounds. (A) Pictures of wild type (WS), clca-2 , complemented line ( CLCa-Ox ( clca-2 )) and NRT2.7 over-expressing line in clca-2 ( NRT2.7-Ox ( clca-2 )) on the left side and, WS and the two NRT2.7 over-expressing lines, NRT2.7-Ox1 (WS) and NRT2.7-Ox2 (WS) on the right side. Plants were grown on 2 mM nitrate for 12 days (Scale bars=1 cm). Shoot fresh weight (B) and total root length (C) were measured on wild type (WS), clca-2 , CLCa-Ox ( clca-2 ) and NRT2.7-Ox ( clca-2 ) (left side of the panel) or WS, NRT2.7-Ox1 (WS) and NRT2.7-Ox2 (WS) (right side of the panel). The number of samples were 4-5 for shoot fresh weight (one sample = a pool of 3-4 plants) and n=15-17 for total root length on individual plants. One representative experiment out of three is shown. The numbers above the bars showed the percentage increase or decrease compared to WS. Presented data are means ± Standard Error of the Mean (SEM). Different letters at the top of the bars represent significant difference (p value<0.05). To study the activity of NRT2.7, we overexpressed ectopically NRT2.7 in the clca-2 background and as control in WS. To produce the different genotypes, we used the NRT2.7 -overexpressing lines NRT2.7-Ox1 ( WS ) and NRT2.7-Ox2 ( WS ) generated in a previous study ( Chopin et al ., 2007 ) and analysed the NRT2.7 expression in aerial part. It was very weakly expressed in WS whereas the level of its mRNA is very high in NRT2.7-Ox1 ( WS ) and NRT2.7-Ox2 ( WS ) ( Supplementary Fig. S3 ). These two lines were crossed with the clca mutant to obtain NRT2.7-Ox1 ( clca-2 ) and NRT2.7-Ox2 ( clca-2 ), expressing consequently NRT2.7 at the same level than in the WS background . NRT2.7-Ox1 ( clca-2 ) and NRT2.7-Ox2 ( clca-2 ) showed similar phenotypes and representative data for one line are presented. For comparison, we included in our studies a clca-2 mutant overexpressing the CLCa cDNA under the control of the strong 35S promoter of the CaMV (referred thereafter as CLCa-Ox ( clca-2) ) ( Wege et al ., 2010 ). In this line, CLCa transcript levels were increased 30-fold compared to WS ( Wege et al ., 2014 ). Download figure Open in new tab Supplementary Fig. S3. Level of NRT2.7 expression in NRT2.7-Ox (WS). Plants were grown on vertical Petri dishes for 12 days on 2 mM external nitrate as described in the ‘Materials and Methods’. RT-qPCR was performed on total RNA extracted from shoot using NRT2.7 and EF1 α specific primers. Data represents the means ± SEM of three biological replicates. Based on the more pronounced differences in growth between clca-2 and WS under non-limiting external N supply, we cultivated the plants on 2 mM nitrate medium for all further analyses using both clca-2 lines overexpressing either CLCa or NRT2.7 compared to the controls WS, NRT2.7 over-expressing lines ( NRT2.7-Ox1 (WS)) and clca-2. Both CLCa and NRT2.7 overexpression restored the clca-2 growth phenotypes for shoot biomass and total root length ( Fig. 1 ). This growth restoration was also observed in both lines overexpressing NRT2.7 in the clca-1 knock-out background and occurred regardless external nitrate availability ( Supplementary Fig. S2A, B ). Wild-type plants overexpressing NRT2.7 showed an important increase of 60% in total root length and no significant difference in shoot fresh weight compared to WT ( Fig. 1B, C ). The growth stimulation observed for NRT2.7 overexpressing lines both in the clca-2 and the WS background, is therefore independent of the clca-2 mutation. The physiological changes triggered by NRT2.7 overexpression in the clca mutants are thus different from the complementation by CLCa while both led to restored growth. NRT2.7 and CLCa overexpression have opposite effects on both nitrate content and nitrate uptake capacities To decipher the underlying mechanism that impact growth of these overexpressing lines, we determined nitrate content in roots and shoots. As described earlier ( Wege et al ., 2010 ), nitrate content of clca-2 was 35 and 28 % lower than WT level in shoots and roots, respectively ( Fig. 2A, C ). Expression of CLCa in the clca-2 mutant restored nitrate levels in shoots and roots, whereas NRT2.7 overexpression in the clca-2 background led to a 53% decrease of the nitrate content when compared with the clca-2 mutant ( Fig. 2A, C ). Likewise, NRT2.7 overexpression does not complement clca-1 phenotype for shoot and root nitrate content regardless the external nitrate concentration for plant growth ( Supplementary Fig. S2C, D ). This showed that the increased growth observed in the NRT2.7-Ox (clca-2) and NRT2.7-Ox (clca-1) lines occurred without restoring nitrate content. Like the effect of NRT2.7 overexpression in the clca-2 mutant background, overexpression of NRT2.7 in wild-type background was accompanied by a slight decrease in nitrate content in roots but no significant modification was observed in shoots ( Fig. 2B, D ). Download figure Open in new tab Fig. 2. Nitrate content of NRT2.7 overexpression in clca-2 and wild-type backgrounds. Wild type (WS), clca-2 , complemented line ( CLCa-Ox ( clca-2 )) and NRT2.7 over-expressing line in clca-2 ( NRT2.7-Ox ( clca-2 )) (A, C) or WS and the two NRT2.7 over-expressing lines, NRT2.7-Ox1 (WS) and NRT2.7-Ox2 (WS) (B, D), were grown on 2 mM nitrate. After 12 days on the medium, shoot (A, B) and root (C, D) nitrate contents were measured. Two to three biological replicates were performed on each genotype, one sample corresponding to a pool of at least 50 plants. One representative experiment out of two is shown. The numbers above the bars showed the percentage increase or decrease compared to WS. Presented data are means ± SEM. Different letters at the top of the bars represent significant difference (p value<0.05). In order to understand the impact on nitrate levels, we investigated how nitrate uptake was affected in these lines. Nitrate uptake was measured using 15 N labelled nitrate at concentration of 0.2 and 6 mM to monitor both high and low affinity transport system activities (HATS and LATS, respectively, this latter activity being obtained as the difference between uptake measurements at 6 mM and 0.2 mM). Figure 3 shows that the HATS activity was decreased by 16% in clca-2 whereas the LATS activity was not significantly different from that of WT. The CLCa-Ox ( clca-2 ) line exhibited the same uptake capabilities for both transport activities as wild-type plants, illustrating a full complementation of the clca-2 mutant phenotype. The NRT2.7-Ox ( clca-2 ) line showed similar HATS activity as the clca-2 knock-out mutant but an increase by 29% of LATS activity ( Fig. 3B, C ). The NRT2.7-Ox (WS) lines displayed comparable profiles but enhanced effect with a 2-fold increased activity of the LATS when compared to WT ( Fig. 3C ). Thus, whereas CLCa overexpression in the clca mutants increased mainly HATS activity compared to WT, NRT2.7 overexpression had the opposite effect, supporting the hypothesis that these two nitrate transporters have different functions in planta when overexpressed ectopically. Download figure Open in new tab Fig. 3. Effect of NRT2.7 overexpression in clca-2 and wild-type backgrounds on nitrate uptake activities. Wild type (WS), clca-2 , complemented line ( CLCa-Ox ( clca-2 )) , NRT2.7 over-expressing line in clca-2 ( NRT2.7-Ox ( clca-2 )) and the two NRT2.7 over-expressing lines, NRT2.7-Ox1 (WS) and NRT2.7-Ox2 (WS), were grown on vertical Petri dishes on a medium containing 2 mM nitrate for 12 days. Uptake experiments consisted of four to five measurements of 15 NO 3 - in roots from seedling that were incubated for 5 min in a liquid medium containing 6 mM (A = HATS+LATS) or 0.2 mM (B = HATS) 15 NO 3 - . The activity of LATS (C) was obtained by subtracting the activity measured at 0.2 mM to the one at 6 mM (see material and methods section for details). Presented data are averages of three independent experiments (n=3-5 by independent experiment). Bars represents means ± SEM. The numbers above the bars showed the percentage increase or decrease compared to WS. Different letters at the top of the bars represent significant difference (p value<0.05). NRT2.7 overexpression leads to increased biomass under limiting and non-limiting conditions Plants stimulate their root growth when their N nutritional status is low ( Jia and von Wirén, 2020 ). Given the important increase of root growth in the NRT2.7-Ox (WS) lines under non-limiting N supply ( Fig. 1 ), we further studied the growth-promoting effect of NRT2.7 overexpression under limiting N supply to better understand the impacts of the changes in nitrate fluxes through the vacuolar membrane. Plants were grown on vertical agar medium containing 0.2 mM nitrate for 12 days. In these conditions, shoot biomass and total root length of NRT2.7-Ox (WS) lines were increased when compared to WT by 49 to 65% and 19 to 33% respectively ( Fig. 4A, B ). Conversely, root nitrate content of the NRT2.7-Ox (WS) lines was dramatically reduced by 75 to 83% compared to WT, whereas shoot nitrate content was not modified ( Fig. 4C, D ). A similar tendency was observed when NRT2.7 was overexpressed in clca KO mutant comparing nitrate content to shoot and root biomass ( Supplementary Fig. S4 ). Download figure Open in new tab Supplementary Fig. S4. Effect of limiting N supply on the phenotype of NRT2.7 over-expressors in clca-2 background. Wild type (WS), clca-2, complemented line ( CLCa-Ox ( clca-2 )) and NRT2.7 over-expressing line in clca-2 ( NRT2.7-Ox ( clca-2 )) were grown in petri dishes for 12 days on 0.2 mM nitrate. Shoot fresh weight (A), total root length (B) and, shoot and root nitrate contents (C, D) were measured as described in Figure 1 and 2 . Data are averages of three to five measurements, except for B, n=12-19 (bars representing the SEM). Different letters show significant differences between genotypes (p<0.05). Download figure Open in new tab Fig. 4. Effect of limiting N supply on the phenotype of NRT2.7 over-expressers. Wild type (WS) and over-expressing lines, NRT2.7-Ox1 (WS) and NRT2.7-Ox2 (WS), were grown in petri dishes for 12 days on 0.2 mM nitrate. Shoot fresh weight (A), total root length (B) shoot and root nitrate contents (C, D) were measured as described in Figure 1 and 2 . C/N ratios, percentages of N and C contents were measured in shoot (E) and root (F) using an elementary analyser. Data are averages of four biological replicates for shoot fresh weight, 12 to 17 for total root length, two to four for nitrate contents and four for C and N contents. The numbers above the bars showed the percentage increase or decrease compared to WS. Different letters show significant differences between genotypes (p<0.05). The carbon to nitrogen (C/N) ratio is an essential indicator that plants perceive to orientate their growth, development and metabolism ( Coruzzi and Zhou, 2001 ). The C/N ratio of NRT2.7-Ox (WS) compared to WT was increased in shoots and roots mainly due to a decrease in total N proportion ( Fig. 4E ). These data suggest that the ectopic expression of NRT2.7 drives nitrate use for biomass production. As no difference in nitrate uptake through the HATS was observed ( Fig. 3 ), the strong decrease in nitrate content could be the consequence of a lower vacuolar storage capacity and/or an increased nitrate flux out of the vacuole as it was previously shown for other vacuolar nitrate transporters ( He et al ., 2017 ; Lu et al ., 2022 ; Hodin et al ., 2023 ; Shi et al ., 2023 ). NRT2.7 overexpression leads to increased nitrate fluxes out of the vacuole In order to associate the observed physiological changes with NRT2.7 functional properties, we used an electrophysiological approach to determine if the over-expression of NRT2.7 modifies the nitrate fluxes across the vacuolar membrane. We took advantage of the NRT2.7-Ox ( clca-2 ) line where such modification would not be masked by CLCa-driven fluxes. We extracted vacuoles from mesophyll protoplasts of the clca-2 and the two NRT2.7-Ox lines ( clca-2 ) and performed patch clamp experiments in whole-vacuole configuration ( Fig. 5 ). In order to maximize the fluxes of nitrate directed from the vacuole to the cytosol and from the cytosol to the vacuole, we performed two independent series of experimentations designed as follow. In both experiment series, we used the non-permeant cation BisTriPropane in order to limit the vacuolar currents mediated by cations. With the aim to highlight the efflux of nitrate from the vacuole (positive ionic currents), in the first series of experiments ( Fig. 5B ), we used bi-ionic conditions with 200 mM nitrate in the vacuole and 19.2 mM chloride in the cytosolic-side solution. The vacuoles extracted from clca-2 knock-outs presented a mean current density of +1.3 ± 0.4 pA/pF at +43 mV and a mean reversal potential (E rev =-8 ± 3 mV) that were comparable to those previously observed in similar ionic conditions ( De Angeli et al ., 2006 ; Wege et al ., 2010 ). Importantly, in the same ionic conditions, vacuoles from NRT2.7-Ox (clca-2) plants presented significantly higher current densities compared to clca-2 currents only at positive vacuolar membrane potentials (from +3 mV to +83 mV, Fig. 5B ). To note, similar results were obtained for both lines as displayed on the figures by vertical or horizontal diamonds. These positive currents are compatible with an increased nitrate efflux capacity of the vacuoles from NRT2.7-Ox (clca-2) plants. In the second set of experiments, we used an ionic configuration suited to highlight nitrate currents directed from the cytosol to the vacuolar lumen. We performed cytosolic-side solution exchange experiments starting from a chloride-based buffer (19.2 mM Cl - pH 7) to a nitrate based buffer (4 mM NO 3 - pH 7) keeping the vacuolar-side buffer constant and with only chloride as a permeant anion (15 mM Cl - pH 5.5) ( Supplementary Fig. S5A and S5B ). Upon the exchange of the cytosolic-side solution a similar increase of the negative current was observed in vacuoles obtained from the tested genotypes. Indeed, the mean current ratios at -60 mV were I NO3 /I Cl =1.84±0.4 in NRT2.7-Ox (clca-2) vacuoles and I NO3 /I Cl =1.77 ± 0.44 in clca-2 vacuole s . Conversely, the current densities in presence of nitrate did not show a significant difference between the genotypes, being I NO3 =-2.4±1 pA/pF at -60 mV in NRT2.7-Ox ( clca-2 ) and I NO3 =-2.7±0.9 pA/pF at -60 mV in clca-2. As the current amplitudes in NRT2.7 overexpressor show a high variability compared to those of the clca-2 mutant, we checked whether the rectification ratios between the currents at +83 mV versus -77 mV and +63 mV versus -57 mV ( Supplementary Fig. S5C and S5D ) were not different between the vacuoles from the different genotypes. The values of these ratios were around -3 and there was no significant difference between the two genotypes. Further no correlation between the rectification ratio (I +83mV /I -77mV ) and current density (I +83mV ) could be identified. This indicates that the high variability of currents obtained in NRT2.7 -Ox ( clca-2 ) is not linked to background leak currents. In conclusion, this second series of experiments indicates that the vacuoles obtained from NRT2.7-Ox ( clca-2 ) do not display an increased capacity of driving NO 3 - fluxes directed to the vacuolar lumen compared to the vacuoles from clca-2 under our experimental conditions. Altogether, the results obtained by patch-clamp experiments suggest that, compared to vacuoles from clca-2 , NRT2.7-Ox ( clca-2 ) plant vacuoles present a higher capacity to drive nitrate fluxes directed into the cytosol but are not modified concerning nitrate influx into the vacuole. Download figure Open in new tab Supplementary Fig. S5. Nitrate influx is not increased in vacuoles from NRT2.7_Ox ( clca2 ) expressing plants compared to vacuoles of clca-2 knock-outs. (A) mean IV curves measured in vacuoles extracted from NRT2.7-Ox ( clca-2 ) (top, n=6) and clca-2 knock-out (bottom, n=6) without nitrate in the vacuolar lumen (15 mM Cl - , pH=5.7, see Materials and methods) and in absence (square) and presence (circle) of cytosolic nitrate (4 mM NO 3 - pH=7). (B) current ratio (I NO3 - /I cl - ) calculated from cytosolic solution exchange experiments at different membrane potentials. Error bars are standard deviation. (C) Rectification rates calculated as I(+83mV)/I(-77mV) (blue, left axis) and I(+63mV)/I(-57mV) (red, right axis). The Rectification rate was calculated from the individual vacuoles extracted from clca-2 knock-out mutant (triangles) and NRT2.7-Ox ( clca-2 ) transgenic line (circles) shown in Fig. 5 . Ionic currents were recorded in presence of nitrate only in the vacuolar lumen (200 mM NO 3 - pH 5.5 in the vacuolar buffer, 19.2 mM Cl - in the cytosolic buffer, see Materials and Methods). Statistical significance between clca-2 knock-out mutant (triangles) and NRT2.7-Ox ( clca-2 ) transgenic line (circles) was calculated with a non-parametrical Mann-Whitney test. (D) Plot showing the rectification rate I(+83mV)/I(-77mV) (blue, left axis) vs the current density of each vacuole of the NRT2.7-Ox ( clca-2 ) transgenic line. The broken line shows a liner regression fit of the data illustrating the absence of correlation between the current intensity recorded in each vacuole and the rectification. Download figure Open in new tab Fig. 5. Nitrate efflux from mesophyll vacuoles is increased in NRT2.7-Ox ( clca-2 ) plants compared to clca-2 knock-outs. (A) Representative whole-vacuole currents measured in vacuoles extracted from clca-2 knock-out mutant (left) and NRT2.7-Ox ( clca-2 ) transgenic line (right) in presence of nitrate only in the vacuolar lumen (200 mM NO 3 - pH 5.5 in the vacuolar buffer, 19.2 mM Cl - in the cytosolic buffer, see Materials and Methods). Currents were evoked in response to 2 s voltage pulses ranging from -77 mV to +83 mV in +20 mV steps, followed by a tail pulse at +33 mV, with a holding potential at -17 mV. (B) Box plot of the mean current density (pA/pF) measured at different positive (left) and negative (right) membrane potentials in vacuoles from NRT2.7-Ox ( clca-2 ) (vertical diamonds line NRT2.7-Ox1 ( clca-2 ), n=11; horizontal diamonds line NRT2.7-Ox2 ( clca-2 ), n=5), and clca-2 knock-out plants, n=10. The ionic conditions were as in (A). Boxes defines the first and the third quartile and the median, the whiskers represent the ninth and the ninety-first percentiles. The two asterisks indicate a statistical significance at 1% level with a Mann-Whitney test. Discussion Recent studies underline the importance of nitrate fluxes through the vacuolar membrane to enhance NUE by increasing efflux of nitrate from vacuole and/or reducing nitrate storage in this latter compartment ( Han et al ., 2016 ; Wang et al ., 2018 ; Hodin et al ., 2023 ; Shi et al ., 2023 ). It is therefore essential to characterize the different vacuolar nitrate transporters and their activities to get a better understanding of their functional specificities. In this report, we analyzed the function of the vacuolar NRT2.7 transporter by introducing its cDNA under the control of the 35S promoter in clca KO mutant or in wild type. The combined results of the electrophysiological analyses and physiological traits of the CLCa and NRT2.7 overexpressing lines strikingly demonstrate that both NRT2.7 and CLCa impact N use but through clearly distinct mechanisms. The NRT2.7 overexpression in the clca-2 mutant bypassed the CLCa-dependent reduced growth via a mechanism that uncoupled growth and nitrate accumulation probably related to modified nitrate efflux from the vacuole. This mechanism, when acting in a wild-type background overexpressing NRT2.7 , led to an increase of biomass production mainly through a higher root biomass. NRT2.7 stimulates nitrate export out of the vacuole when overexpressed ectopically In this study, we used the CLCa mutant altered physiology to assess functions and physiological consequences of NRT2.7 ectopic expression. We chose CLCa over its closest homologous, CLCb, as the clca KO mutant has a stronger phenotype than clcb in respect to nitrate contents ( Geelen et al ., 2000 ; von der Fecht-Bartenbach et al ., 2010 ; Shi et al ., 2023 ) and, nitrate currents are very small in clca even when CLCb is over-expressed into this mutant compared to wild type ( De Angeli et al ., 2006 ; Monachello et al ., 2009 ). Furthermore, similar to CLCb, CLCa is involved in nitrate efflux out the vacuole depending on its phosphorylation state ( Wege et al ., 2014 ). As the phosphorylation site is conserved in CLCb, we can assume that CLCb could work in both ways depending on the growth conditions. In this study, we confirmed that CLCa mutant physiology was deeply affected by its limiting capacity of loading nitrate into vacuoles ( Fig. 1 and 2 ; Supplementary Fig. S1 and S2 ). Besides CLCa, the NRT2.7 nitrate transporter is located at the tonoplast and has been showed to translocate nitrate in Xenopus oocytes. Its loss-of-function reduces nitrate accumulation in seeds where the gene is chiefly expressed ( Chopin et al ., 2007 ). Although NRT2.7 mRNA were detected in leaves and roots, no phenotype other than seed traits have been reported in the KO mutant ( Orsel et al ., 2002 ; Okamoto et al ., 2003 ; Chopin et al ., 2007 ). To better characterize its function in planta , we overexpressed NRT2.7 in wild type and clca KO mutant backgrounds. The overexpression of NRT2.7 fully restores the reduced clca-2 plant growth and even exceeds wild-type biomass but, surprisingly, further diminished the low clca-2 nitrate content ( Fig. 1 and 2 ). It led to 35% and 23% decreases of shoot and root nitrate pools, respectively, when compared to clca-2 . These results showed that NRT2.7 could indeed rescue the clca-2 growth defect but rather through an alternative mechanism than a true functional complementation. This hypothesis was confirmed as the NRT2.7 overexpressors in both wild-type and clca-2 background showed an increase in total root length and a depletion of root nitrate content compared to wild-type plants. The decrease of root nitrate content caused by NRT2.7 ectopic overexpression is difficult to conceive with a vacuolar nitrate importer. Chopin et al . (2007) reported an influx of 15 N labeled nitrate into NRT2.7 expressing oocytes. This result indicates that, in this biological system, NRT2.7 was targeted to the plasma membrane with presumably its cytoplasmic N- and C-terminal domains oriented to the oocyte cytoplasm. In planta , NRT2.7 is localized at the tonoplast ( Chopin et al ., 2007 ). As NRT2.7 cytoplasmic terminus is probably maintained in the plant cell cytoplasm ( Jacquot et al ., 2020 ), NRT2.7 would thus function as a nitrate efflux system from the vacuole to the cytoplasm. This orientation is in agreement with the fact that NRT2s are H + /NO 3 - symporters ( Forde and Clarkson, 1999 ) and that roots of NRT2.7 overexpressors become depleted of nitrate at low external nitrate availability ( Fig. 4 , Supplementary Fig. S4 ). In addition, the determination of nitrate fluxes by patch clamp techniques showed that isolated vacuoles from NRT2.7-Ox ( clca-2 ) plants displayed a higher capacity to drive nitrate fluxes directed to the cytosol th an isolated vacuoles from clca-2 plants ( Fig. 5 ). The difference of current density between the two genotypes is rather small (i.e about 1.3 pA/pF more in NRT2.7-Ox ( clca-2 ) vacuoles at +63 mV). This can be explained by a difference of electrogenicity and unitary turnover rate between the two kinds of ion transporters, CLCa being a nitrate/proton antiporter and NRT2.7 a nitrate/proton symporter ( Forde and Clarkson, 1999 ; De Angeli et al ., 2006 ). In the same experimental setup, we showed that the capacity of such isolated vacuoles to drive nitrate influx did not vary between these genotypes ( Fig. 5 and Supplementary Fig. S5 ). This indicates that NRT2.7 stimulates vacuolar nitrate export when ectopically overexpressed in seedlings and could explain the observed decrease in nitrate content when NRT2.7 is overexpressed in clca-2 background. As this latter effect was also detected in NRT2.7-Ox (WS) mainly when the plants were grown on 0.2 mM nitrate ( Fig. 4 and Supplementary Fig. S4 ), the activity of NRT2.7 to stimulate nitrate export does not seem to rely on the import activity of CLCa. Additionally, based on these experiments, we cannot completely exclude that NRT2.7 may regulate positively the activity of nitrate exporters localized in the tonoplast and is not itself the active transporter driving the fluxes. CLCb , recently shown to be a nitrate exporter when nitrate is low in the external medium and, overexpressed in clca KO mutant ( Monachello et al ., 2009 ; Shi et al ., 2023 ), could have its activity induced by NRT2.7 overexpression. However, the decrease of root nitrate content by -75 and -83% in NRT2.7-Ox (WS) compared to wild type was accompanied by a total root growth increase of 33% when plants are grown on 0.2 mM nitrate ( Fig. 4 ). Under the same growth conditions, a similar tendency was observed in NRT2.7-Ox ( clca2), a 19% increase in root length was accompanied by a 84% reduction in root nitrate content ( Supplementary Fig. S4 ). As CLCb is over-expressed in clca mutant ( Monachello et al ., 2009 ), this that CLCb might not be involved in the stimulation of nitrate export by NRT2.7 overexpression. Chopin et al . (2007) observed a decrease in seed nitrate content in nrt2.7 mutants, which was interpreted as a reduced capacity to import nitrate into the vacuole of the embryo. NRT2.7 is highly expressed in seeds and the NRT2.7 promoter drives expression in the embryos where the protein is located at the tonoplast. A decreased nitrate content is thus difficult to explain by the loss-of-function of a vacuolar nitrate exporter protein. However, the transport direction of NRT2.7 in seeds and in shoot/root vacuoles might be different because of distinct types of vacuoles existing in these organs ( Shimada et al ., 2018 ). In addition, tonoplast membrane potential might vary and the transport direction may reflect the organ specific ionic and osmotic conditions as it has been discussed previously for NPF6.3 (NRT1.1/CHL1, ( Léran et al ., 2014 ) or CLCa ( Wege et al ., 2014 ). Besides, nitrate transport properties are regulated via posttranslational protein modifications (e.g. Wege et al ., 2014 ; Jacquot et al ., 2017 ), a mechanism that should be explored in the future for NRT2.7. However, no phosphorylation site in NRT2.7 is found in the PhosPhAt databases ( Heazlewood et al ., 2008 ). Finally, another hypothesis would be that nrt2.7 seed nitrate content is decreased because nitrate transport from the roots or shoots towards the seeds is diminished in the nrt2.7 mutants. Indeed, in Arabidopsis seeds, nitrate content seems to rely directly on the nitrate taken up by roots ( Schulze et al ., 1994 ). Inflorescence grafting experiments are required to distinguish between seed prone or root/shoot prone determination of the decreased nitrate level in nrt2.7 seeds. Altogether, these results support that NRT2.7 is essential for nitrate fluxes through the vacuolar membrane. It stimulates nitrate export from the vacuole in seedlings but the contrasting phenotype in reproductive and vegetative organs when the expression of NRT2.7 is modified, poses a challenging hypothesis and needs further investigation. Nitrate fluxes through CLCa and NRT2.7 alter root nitrate uptake As previously reported, we confirmed that the mutation in CLCa reduced nitrate uptake activity in the high affinity range ( Monachello et al ., 2009 , Fig. 3 ) whereas the level of LATS activity was not modified. These results illustrate the existence of a coordinated signaling crosstalk between vacuolar and plasma membrane nitrate fluxes. An obvious signal for this coordinated transmembrane transport would be the nitrate anion itself. It was shown that loss of function of CLCa leads to a faster increase of the cytosolic nitrate concentration in response to an increase of the extracellular NO 3 - concentration in guard cells ( Demes et al ., 2020 ). In this scenario, a transitory elevated cytosolic nitrate level would decrease nitrate uptake, increase the nitrate efflux from the cells and/or the assimilation pathways through the nitrate reductase (NR). This was confirmed in a recent study demonstrating that the inhibition of nitrate storage in the vacuole leads to an increase of the NR activity and a better NUE ( Hodin et al ., 2023 ). In addition to nitrate, nutrient uptake activity is controlled by a wide range of biophysical (membrane potential, pH) or physiological signals (phosphorylation, metabolites) (see Amtmann and Blatt, 2009 ). CLCa through its activity as a NO 3 - /H + exchanger is known for its function in adjusting the cytosolic pH in guard cells ( Demes et al ., 2020 ). More recently, Jain and Schmidt (2024) showed that NRT2.1 phosphorylation level varies according to the environmental pH certainly to adjust cytosolic pH. Then the loss of function of CLCa could lead to the inactivation of NRT2.1 to compensate the change in cytosolic pH. The increase of nitrate export from the vacuole due to the NRT2.7 overexpression leads probably to change in nitrate cytosolic content and/or cytosolic pH, as for clca-2 . Even if this hypothesis needs to be tested using the nitrate/pH biosensor, ClopHensor ( Demes et al ., 2020 ), these modifications could explain the decrease in HATS activity observed in NRT2.7-Ox (clca-2) like in clca-2 plants but not the increase of the LATS activity ( Fig. 3 ). This latter discrepancy could suggest that alterations in nitrate influx and efflux in and out of the vacuole do not have the same effect on nitrate uptake. The coordination between the direction of vacuolar nitrate fluxes and nitrate influx at the plasma membrane will need to be further studied in the future. NRT2.7 overexpression increases growth at low external and internal nitrate The overexpression of NRT2.7 stimulates nitrate export from the vacuole, leading to an increase in plant growth. This phenotype could be explained not only by the increase of LATS activity but also by a higher nitrate long-distance transport from root to shoot. A study characterizing two Brassica napus genotypes differing by their capacity in nitrate storage in root previously showed that the decrease of root nitrate content induces a higher nitrate translocation from root to shoot leading to a better NUE ( Han et al ., 2016 ). We can assume that a similar phenomenon occurred in the NRT2.7 overexpressors. Interestingly, both shoot and root biomass increased in NRT2.7-Ox (WS) grown under limiting N nutrition even that a severe decrease of nitrate in root (-75 to -83% compared to wild type) and a slight decrease of total nitrogen content (-8 to –19% compared to wild type) occurred in these growth conditions ( Fig. 4 ). These results confirm an apparent disconnection between growth and nitrate content in NRT2.7 overexpressors plants. C and N metabolism are interconnected and regulate each other ( Zheng, 2009 ). Interestingly, the C/N ratio was higher in the overexpressors compared to wild type due to a decrease in N proportion indicating that, even if plants are grown under low external N availability, the overexpressors are able to maintain C proportions by regulating photosynthesis activity facilitating the increase in biomass. The low internal nitrate levels could be then the consequence of N dilution occurring under accelerated growth. At the opposite, the increase of nitrate export from the vacuole could stimulate nitrate assimilation and consequently photosynthesis as shown in a Brassica napus genotype with reduced root nitrate storage ( Han et al ., 2016 ) or, more recently when Arabidopsis plants overexpressing the CLCb nitrate vacuolar exporter are grown on low nitrate ( Shi et al ., 2023 ). In conclusion, we show that the two vacuolar nitrate transporters CLCa and NRT2.7 share limiting functionality and are not exchangeable. Our results suggest that NRT2.7 stimulates vacuolar nitrate export when overexpressed ectopically. By exploiting genotypes with modified levels of vacuolar transporters, we show that changes in the nitrate influx or efflux from the vacuolar compartment lead to differences in root nitrate uptake suggesting the existence of a fine regulation between vacuolar and plasma membranes through biophysical and physiological networks of vacuolar nitrate sensing and homeostasis. NRT2.7, whose function in vivo seemed to be restricted to seeds, triggers new physiological features when ectopically overexpressed. The link between NRT2.7-driven nitrate fluxes and C content requires further studies but may be the basis for improving plants with higher C/N ratios accompanied by increased growth when N is limiting. Supplementary data The following supplementary data are available at JXB online. Fig. S1. Growth and nitrate content of clca-2 knock-out mutant grown on different nitrate concentrations. Fig. S2. Growth and nitrate content of NRT2.7 overexpressors in the c lca-1 knock-out mutant. Fig. S3. Level of NRT2.7 expression in NRT2.7-Ox (WS) lines. Fig. S4. Effect of limiting N supply on the phenotype of NRT2.7 over-expressors in clca-2 background. Fig. S5. Nitrate influx in vacuoles from NRT2.7 -Ox ( clca-2 ) plants and from clca-2 knock-out. Author contributions FDV, AK and SF : conceptualization design ; PA, FDV, ADA, AK and SF: experimental design ; PA, ADA, PB and VB performed the experimental work ; PA, MD, FDV, ADA, AK and SF analyzed the data; PA, FDV, AK and SF wrote the manuscript. All authors reviewed the manuscript. Conflict of interest The authors declare no conflict of interest. Funding This work benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) and of the Agence Nationale de la Recherche (ANR-Nitrapool ANR08-Blan-008). Data availability All the raw data supporting the findings of this work are available upon request from the corresponding authors, Anne Krapp and Sophie Filleur. Statistical analyses are available in the supplementary table 1. Abbreviations CaMV Cauliflower Mosaic Virus CLC ChLoride Channel KO Knock-out N Nitrogen NPF Nitrate transporter 1/Peptide transporter Family NRT2 High affinity Nitrate transporter NUE Nitrogen Use Efficiency WS Wassilewskija Arabidopsis accession Acknowledgements The authors are very grateful to Pascal Tillard for servicing the 15 N measurements, Anne Marmagne for helping with the elementary C/N analyzer and the IJPB platform ‘The Plant Observatory’, in particular Joël Talbotec and Philippe Maréchal for taking care of greenhouse cultures. Footnotes ↵ * INRIA Rocquencourt, Domaine de Voluceau, Rocquencourt - BP105, 78153 Le Chesnay Cedex, France ↵ ** Institute for Plant Sciences of Montpellier (IPSiM), Université de Montpellier, CNRS, INRAE, Institut Agro, Montpellier, 34060, France. ↵ *** Biomolécules et Biotechnologies Végétales, EA2106, Université de Tours, Tours, France Email addresses of the authors : Patrick Armengaud : patrick.armengaud{at}inria.fr Alexis De Angeli : alexis.deangeli{at}cnrs.fr Patrick Berquin : patrick.berquin{at}inrae.fr Virginie Bréhaut : virginie.brehaut{at}inrae.fr Mickaël Durand : midurand{at}univ-tours.fr Françoise Daniel-Vedele : fravedele{at}yahoo.fr Figures revised: data not normalized anymore Supplemental figure S4 added. Supplemental figure S5 revised. Presisions added throughout the text. References 1. ↵ Amtmann A , Blatt MR . 2009 . Regulation of macronutrient transport . The New Phytologist 181 , 35 – 52 . OpenUrl CrossRef PubMed 2. ↵ Armengaud P , Zambaux K , Hills A , Sulpice R , Pattison RJ , Blatt MR , Amtmann A . 2009 . EZ-Rhizo: integrated software for the fast and accurate measurement of root system architecture . The Plant Journal: For Cell and Molecular Biology 57 , 945 – 956 . OpenUrl CrossRef 3. ↵ Barbier-Brygoo H , De Angeli A , Filleur S , Frachisse J-M , Gambale F , Thomine S , Wege S . 2011 . Anion channels/transporters in plants: from molecular bases to regulatory networks . Annual Review of Plant Biology 62 , 25 – 51 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Bergsdorf E-Y , Zdebik AA , Jentsch TJ . 2009 . Residues important for nitrate/proton coupling in plant and mammalian CLC transporters . The Journal of Biological Chemistry 284 , 11184 – 11193 . OpenUrl Abstract / FREE Full Text 5. ↵ Cassman KG , Dobermann A , Walters DT . 2002 . Agroecosystems, nitrogen-use efficiency, and nitrogen management . Ambio 31 , 132 – 140 . OpenUrl PubMed 6. ↵ Chopin F , Orsel M , Dorbe M-F , Chardon F , Truong H-N , Miller AJ , Krapp A , Daniel-Vedele F . 2007 . The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds . The Plant Cell 19 , 1590 – 1602 . OpenUrl Abstract / FREE Full Text 7. ↵ Cookson SJ , Williams LE , Miller AJ . 2005 . Light-dark changes in cytosolic nitrate pools depend on nitrate reductase activity in Arabidopsis leaf cells . Plant Physiology 138 , 1097 – 1105 . OpenUrl Abstract / FREE Full Text 8. ↵ Coruzzi GM , Zhou L . 2001 . Carbon and nitrogen sensing and signaling in plants: emerging ‘matrix effects’ . Current Opinion in Plant Biology 4 , 247 – 253 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Cross JM , von Korff M , Altmann T , Bartzetko L , Sulpice R , Gibon Y , Palacios N , Stitt M . 2006 . Variation of enzyme activities and metabolite levels in 24 Arabidopsis accessions growing in carbon-limited conditions . Plant Physiology 142 , 1574 – 1588 . OpenUrl Abstract / FREE Full Text 10. ↵ De Angeli A , Monachello D , Ephritikhine G , Frachisse JM , Thomine S , Gambale F , Barbier-Brygoo H . 2006 . The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles . Nature 442 , 939 – 942 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Dechorgnat J , Nguyen CT , Armengaud P , Jossier M , Diatloff E , Filleur S , Daniel-Vedele F . 2011 . From the soil to the seeds: the long journey of nitrate in plants . Journal of Experimental Botany 62 , 1349 – 1359 . OpenUrl CrossRef PubMed Web of Science 12. ↵ Demes E , Besse L , Cubero-Font P , Satiat-Jeunemaitre B , Thomine S , De Angeli A . 2020 . Dynamic measurement of cytosolic pH and [NO3 -] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis . Proceedings of the National Academy of Sciences of the United States of America 117 , 15343 – 15353 . OpenUrl Abstract / FREE Full Text 13. ↵ von der Fecht-Bartenbach J , Bogner M , Dynowski M , Ludewig U . 2010 . CLC-b-mediated NO-3/H+ exchange across the tonoplast of Arabidopsis vacuoles . Plant & Cell Physiology 51 , 960 – 968 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Forde BG , Clarkson DT . 1999 . Nitrate and Ammonium Nutrition of Plants: Physiological and Molecular Perspectives . In: Callow JA , ed. Advances in Botanical Research . Academic Press , 1 – 90 . 15. ↵ Geelen D , Lurin C , Bouchez D , Frachisse JM , Lelièvre F , Courtial B , Barbier-Brygoo H , Maurel C . 2000 . Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content . The Plant Journal: For Cell and Molecular Biology 21 , 259 – 267 . OpenUrl CrossRef 16. ↵ Govindasamy P , Muthusamy SK , Bagavathiannan M , et al. 2023 . Nitrogen use efficiency-a key to enhance crop productivity under a changing climate . Frontiers in Plant Science 14 , 1121073 . OpenUrl CrossRef PubMed 17. ↵ Han Y-L , Song H-X , Liao Q , et al. 2016 . Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus . Plant Physiology 170 , 1684 – 1698 . OpenUrl Abstract / FREE Full Text 18. ↵ He Y-N , Peng J-S , Cai Y , Liu D-F , Guan Y , Yi H-Y , Gong J-M . 2017 . Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis . Scientific Reports 7 , 6417 . OpenUrl CrossRef PubMed 19. ↵ Heazlewood JL , Durek P , Hummel J , Selbig J , Weckwerth W , Walther D , Schulze WX . 2008 . PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor . Nucleic Acids Research 36 , D1015 – 1021 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Hodin J , Lind C , Marmagne A , et al. 2023 . Proton exchange by the vacuolar nitrate transporter CLCa is required for plant growth and nitrogen use efficiency . The Plant Cell 35 , 318 – 335 . OpenUrl CrossRef PubMed 21. ↵ Howarth RW , Marino R . 2006 . Nitrogen as the limiting nutrient for eutrophication in coastal marine ecosystems: Evolving views over three decades . Limnology and Oceanography 51 , 364 – 376 . OpenUrl CrossRef GeoRef Web of Science 22. ↵ Hu R , Qiu D , Chen Y , Miller AJ , Fan X , Pan X , Zhang M . 2016 . Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply . Frontiers in Plant Science 7 , 1529 . OpenUrl PubMed 23. ↵ Jacquot A , Chaput V , Mauries A , et al. 2020 . NRT2.1 C-terminus phosphorylation prevents root high affinity nitrate uptake activity in Arabidopsis thaliana . The New Phytologist 228 , 1038 – 1054 . OpenUrl CrossRef PubMed 24. ↵ Jacquot A , Li Z , Gojon A , Schulze W , Lejay L . 2017 . Post-translational regulation of nitrogen transporters in plants and microorganisms . Journal of Experimental Botany 68 , 2567 – 2580 . OpenUrl CrossRef PubMed 25. ↵ Jain D , Schmidt W . 2024 . Protein Phosphorylation Orchestrates Acclimations of Arabidopsis Plants to Environmental pH . Molecular & cellular proteomics: MCP 23 , 100685 . OpenUrl CrossRef PubMed 26. ↵ Jia Z , von Wirén N . 2020 . Signaling pathways underlying nitrogen-dependent changes in root system architecture: from model to crop species . Journal of Experimental Botany 71 , 4393 – 4404 . OpenUrl CrossRef PubMed 27. ↵ Kiba T , Feria-Bourrellier A-B , Lafouge F , et al. 2012 . The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants . The Plant Cell 24 , 245 – 258 . OpenUrl Abstract / FREE Full Text 28. ↵ van der Leij M , Smith SJ , Miller AJ . 1998 . Remobilisation of vacuolar stored nitrate in barley root cells . Planta 205 , 64 – 72 . OpenUrl CrossRef Web of Science 29. ↵ Léran S , Varala K , Boyer J-C , et al. 2014 . A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants . Trends in Plant Science 19 , 5 – 9 . OpenUrl CrossRef PubMed Web of Science 30. ↵ Lezhneva L , Kiba T , Feria-Bourrellier A-B , Lafouge F , Boutet-Mercey S , Zoufan P , Sakakibara H , Daniel-Vedele F , Krapp A . 2014 . The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants . The Plant Journal: For Cell and Molecular Biology 80 , 230 – 241 . OpenUrl CrossRef 31. ↵ Liu Q , Wu K , Song W , Zhong N , Wu Y , Fu X . 2022 . Improving Crop Nitrogen Use Efficiency Toward Sustainable Green Revolution . Annual Review of Plant Biology 73 , 523 – 551 . OpenUrl CrossRef PubMed 32. ↵ Lu Y-T , Liu D-F , Wen T-T , Fang Z-J , Chen S-Y , Li H , Gong J-M . 2022 . Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana . Frontiers in Plant Science 13 , 926809 . OpenUrl CrossRef PubMed 33. Matt P , Geiger M , Walch-Liu P , Engels C , Krapp A , Stitt M . 2001 . The immediate cause of the diurnal changes of nitrogen metabolism in leaves of nitrate-replete tobacco: a major imbalance between the rate of nitrate reduction and the rates of nitrate uptake and ammonium metabolism during the first part of the light period. Plant , Cell & Environment 24 , 177 – 190 . OpenUrl CrossRef 34. ↵ McIntyre GI . 1997 . The role of nitrate in the osmotic and nutritional control of plant development . Australian Journal of Plant Physiology 24 , 103 – 118 . OpenUrl CrossRef Web of Science 35. ↵ Melino VJ , Tester MA , Okamoto M . 2022 . Strategies for engineering improved nitrogen use efficiency in crop plants via redistribution and recycling of organic nitrogen . Current Opinion in Biotechnology 73 , 263 – 269 . OpenUrl CrossRef PubMed 36. ↵ Miller AJ , Fan X , Orsel M , Smith SJ , Wells DM . 2007 . Nitrate transport and signalling . Journal of Experimental Botany 58 , 2297 – 2306 . OpenUrl CrossRef PubMed Web of Science 37. ↵ Miranda KM , Espey MG , Wink DA . 2001 . A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite . Nitric Oxide: Biology and Chemistry 5 , 62 – 71 . OpenUrl CrossRef Web of Science 38. ↵ Monachello D , Allot M , Oliva S , Krapp A , Daniel-Vedele F , Barbier-Brygoo H , Ephritikhine G . 2009 . Two anion transporters AtClCa and AtClCe fulfil interconnecting but not redundant roles in nitrate assimilation pathways . The New Phytologist 183 , 88 – 94 . OpenUrl CrossRef PubMed 39. ↵ Neher E . 1992 . Correction for liquid junction potentials in patch clamp experiments. Ion Channels . Methods in Enzymology . Academic Press , 123 – 131 . 40. ↵ Okamoto M , Vidmar JJ , Glass ADM . 2003 . Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision . Plant & Cell Physiology 44 , 304 – 317 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Orsel M , Eulenburg K , Krapp A , Daniel-Vedele F . 2004 . Disruption of the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth at low external nitrate concentration . Planta 219 , 714 – 721 . OpenUrl CrossRef PubMed Web of Science 42. ↵ Orsel M , Filleur S , Fraisier V , Daniel-Vedele F . 2002 . Nitrate transport in plants: which gene and which control? Journal of Experimental Botany 53 , 825 – 833 . OpenUrl CrossRef PubMed Web of Science 43. ↵ Ow DW , Jacobs JD , Howell SH . 1987 . Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity . Proceedings of the National Academy of Sciences of the United States of America 84 , 4870 – 4874 . OpenUrl Abstract / FREE Full Text 44. ↵ Schulze W , Schulze E-D , Stadler J , Heilmeier H , Stitt M , Mooney H a. 1994 . Growth and reproduction of Arabidopsis thaliana in relation to storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptake-deficient mutants. Plant , Cell & Environment 17 , 795 – 809 . OpenUrl CrossRef 45. ↵ Shi Y , Liu D , He Y , Tang J , Chen H , Gong P , Luo J-S , Zhang Z . 2023 . CHLORIDE CHANNEL-b mediates vacuolar nitrate efflux to improve low nitrogen adaptation in Arabidopsis. Plant Physiology , kiad 438 . 46. ↵ Shimada T , Takagi J , Ichino T , Shirakawa M , Hara-Nishimura I . 2018 . Plant Vacuoles . Annual Review of Plant Biology 69 , 123 – 145 . OpenUrl CrossRef PubMed 47. ↵ Wang W , Hu B , Yuan D , et al. 2018 . Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice . The Plant Cell 30 , 638 – 651 . OpenUrl Abstract / FREE Full Text 48. ↵ Wege S , De Angeli A , Droillard M-J , et al. 2014 . Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid . Science Signaling 7 , ra65 . OpenUrl Abstract / FREE Full Text 49. ↵ Wege S , Jossier M , Filleur S , Thomine S , Barbier-Brygoo H , Gambale F , De Angeli A . 2010 . The proline 160 in the selectivity filter of the Arabidopsis NO(3)(-)/H(+) exchanger AtCLCa is essential for nitrate accumulation in planta . The Plant Journal: For Cell and Molecular Biology 63 , 861 – 869 . OpenUrl CrossRef 50. ↵ Zheng Z-L . 2009 . Carbon and nitrogen nutrient balance signaling in plants . Plant Signaling & Behavior 4 , 584 – 591 . OpenUrl CrossRef PubMed Back to top Previous Next Posted February 13, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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