Ying Liu

Rodolfo A Maniero

Ricardo F H Giehl

Michael Melzer

Priscille Steensma

Gabriel Krouk

Teresa B Fitzpatrick

Nicolaus Von Wirén
email: vonwiren@ipk-gatersleben.de

PDX1.1-dependent biosynthesis of vitamin B 6 protects roots from ammoniuminduced oxidative stress

Keywords: ammonium nutrition, apoplastic pH, Fe mobilization, root elongation, pyridoxine, ROS scavenging

Ammonium supply triggers a localized Fe-dependent oxidative burst that arrests primary root elongation but also induces PDX1.1-dependent vitamin B6 production to quench ammonium-induced ROS formation.

INTRODUCTION

Nitrogen (N) is an essential mineral element for plant development and extensively applied in crop production [START_REF] Xu | Plant nitrogen assimilation and use efficiency[END_REF]. While synthetic N fertilizers greatly improve global crop yield, the input of nitrate-based N fertilizers also bears environmental risks when leached as nitrate or emitted as nitrogen oxide [START_REF] Ju | Reducing environmental risk by improving N management in intensive Chinese agricultural systems[END_REF][START_REF] Sutton | Too much of a good thing[END_REF]. Since ammonium is less prone to leaching than nitrate, ammonium-based N fertilizers are widely used nowadays to replace nitrate in agricultural plant production. To increase fertilizer use efficiency, ammonium is often supplied in locally restricted fertilizer strips, where it is present at very high concentrations [START_REF] Nkebiwe | Fertilizer placement to improve crop nutrient acquisition and yield: A review and metaanalysis[END_REF]. Although ammonium is a preferential inorganic N source for many plant species [START_REF] Gazzarrini | Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into arabidopsis roots[END_REF], excessive ammonium causes toxicity resulting in leaf chlorosis and suppressed root growth [START_REF] Britto | NH4 + toxicity in higher plants: a critical review[END_REF]. These symptoms rely on ammonium triggering multiple physiological and morphological responses, including changes in apoplastic and cytosolic pH, gene expression, protein modification, ion transport, N metabolism, redox and phytohormone status, as well as root system architecture [START_REF] Britto | NH4 + toxicity in higher plants: a critical review[END_REF][START_REF] Li | Ammonium stress in Arabidopsis: signaling, genetic loci, and physiological targets[END_REF]Liu and von Wirén, 2017). Against this background and with the increasing use of ammonium-based N fertilizers in agricultural nutrient management, it is important to obtain a deeper understanding of how roots adapt to an ammonium-replete environment.

The most typical morphological changes in root system architecture of ammoniumexposed roots are arrested elongation of primary and lateral roots and enhanced branching of lateral roots (Li et al., 2010;[START_REF] Lima | Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3dependent manner[END_REF][START_REF] Liu | Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana[END_REF].

Regarding lateral root branching, it has recently been shown that apoplastic acidification caused by AMMONIUM TRANSPORTER (AMT)-mediated ammonium uptake provokes pH-dependent radial auxin diffusion to stimulate locally the emergence of lateral root primordia, forming a highly branched root system [START_REF] Meier | Auxinmediated root branching is determined by the form of available nitrogen[END_REF]. Given that in several auxin-related mutants root elongation is still as sensitive to ammonium as in wild-type plants, the repression of root elongation by ammonium is unlikely due to altered distribution or action of auxin in plants (Li et al., 2010;[START_REF] Liu | Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana[END_REF]. Thus, altered root elongation and lateral root branching under ammonium supply are most likely governed by distinct mechanisms. Neither root elongation nor lateral root branching can be triggered by glutamine, the primary assimilation product of ammonium [START_REF] Lima | Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3dependent manner[END_REF][START_REF] Rogato | Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus[END_REF], J o u r n a l P r e -p r o o f suggesting that ammonium itself or ammonium-related metabolites or signals are critical for root development (Liu and von Wirén, 2017). Indeed, excessive ammonium accumulation and hypersensitive root growth of the cipk23 mutant indicated that ammonium-induced calcium signaling is required to re-adjust internal NH4 + /K + ratios [START_REF] Shi | NH4 + toxicity, which is mainly determined by the high NH4 + /K + ratio, is alleviated by CIPK23 in Arabidopsis[END_REF]. This refers to the function of CIPK23, a CBLinteracting protein kinase, in modulating AMT-, HAK-and AKT-type transporter activities [START_REF] Ragel | The CBL-interacting protein kinase CIPK23 regulates HAK5-mediated high-affinity K + uptake in Arabidopsis roots[END_REF][START_REF] Straub | The kinase CIPK23 inhibits ammonium transport in Arabidopsis thaliana[END_REF][START_REF] Sanchez-Barrena | Recognition and activation of the plant AKT1 potassium channel by the kinase CIPK23[END_REF][START_REF] Dong | Emerging roles of the CBL-CIPK calcium signaling network as key regulatory hub in plant nutrition[END_REF] with strong impact on cytosolic ammonium accumulation and root growth.

A further ammonium-related signal may be ethylene, which appears to modulate genes involved in ammonium uptake and assimilation via the transceptor function of NRT1.1 [START_REF] Jian | NRT1.1-related NH4 + toxicity is associated with a disturbed balance between NH4 + uptake and assimilation[END_REF]. Even though these signaling processes help maintaining cytosolic ammonium homeostasis, they do not explain ammonium-dependent root growth inhibition.

Horizontal split agar experiments indicate that the critical region to sense the inhibitory effect of ammonium on root elongation is the root tip (Li et al., 2010). At the cellular level, both cell division and cell elongation are substantially repressed by ammonium, while the integrity of the root stem cell niche remains unaffected [START_REF] Liu | Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana[END_REF]. On the search for mutants overcoming ammonium-induced root growth inhibition, the vtc1-1 mutant was isolated, which is defective in GMP-mannose pyrophosphorylase (GMPase) and thus not only in downstream ascorbate biosynthesis but also in N-glycosylation of proteins [START_REF] Qin | GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana[END_REF]. However, despite its significant impact on ammonium fluxes (Li et al., 2010), GMPase activity turned out not to be the primary cause of ammonium-dependent root growth inhibition [START_REF] Kempinski | Toward the mechanism of NH4 + sensitivity mediated by Arabidopsis GDP-mannose pyrophosphorylase[END_REF]. Transcriptome studies have indicated that enhanced formation of ROS is a typical ammonium-related physiological response [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF]. Even though significantly higher steady-state H2O2 levels in roots were not detected, antioxidative defense systems were induced that comprise ROS-degrading enzymes, including superoxide dismutase, catalase, peroxidase, guaiacol peroxidase and glutathione reductase [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF]. However, in leaves ammonium toxicity increased H2O2 formation, which was found to be mediated by ethylene and followed by enhanced peroxidase activity probably via ABA signaling [START_REF] Li | The Arabidopsis AMOT1/EIN3 gene plays an important role in the amelioration of ammonium toxicity[END_REF][START_REF] Sun | Endogenous ABA alleviates rice ammonium toxicity by reducing ROS and free J o u r n a l P r e -p r o o f ammonium via regulation of the SAPK9-bZIP20 pathway[END_REF]. Also in the roots of rice, ammonium treatment increased H2O2 contents and induced the heme-heme oxygenase OsSE5 together with other ROSdetoxifying enzymes including superoxide dismutase, catalase, and ascorbate J o u r n a l P r e -p r o o f peroxidase to relieve ammonium-supplied plants from oxidative stress [START_REF] Xie | Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa[END_REF]. These studies indicate that ammonium toxicity causes ROS formation and that the capacity of enzymatic ROS detoxification is a major determinant of ammonium tolerance in plants. So far, evidence for the involvement of non-enzymatic antioxidants in ammonium-induced oxidative stress defense was not obtained, considering that in roots the pool sizes and oxidative states of NADH, NADPH, glutathione and ascorbate were not substantially altered by ammonium supply [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF].

To explore the mechanisms by which ammonium-exposed roots cope with ammonium toxicity and ammonium-dependent ROS formation, we first used a pharmacological approach unraveling localization of the ammonium-triggered H2O2 burst. We then screened mutants of ammonium-responsive genes in roots and identified PDX1.1, a gene involved in the biosynthesis de novo of vitamin B6.

Chemical complementation and genetic approaches showed that nonphosphorylated forms of vitamin B6 suppressed H2O2 formation under ammonium supply. With PDX1.1-dependent vitamin B6 formation, our study identifies a protective mechanism that spatially overlaps with ammonium-triggered H2O2 formation in inner root cells and thus carries potential to better adapt plant roots to ammonium-based fertilization strategies.

RESULTS

Accumulation of H2O2 under ammonium supply inhibits primary root elongation

When wild-type seedlings of Arabidopsis were grown on half-strength MS medium for 6 d in the presence of either 1 or 10 mM ammonium as the sole N source, primary root length became 25 or 35% shorter, respectively, than that of nitrate-grown plants (Fig S1A,B). Likewise, total and mean length of lateral roots were repressed to a similar extent as primary root length, suggesting a common mechanism underlying ammonium-induced growth inhibition in both root types . A significant dose-dependent decrease in root elongation rate set in already after 1 d of ammonium exposure suggesting a rapid inhibitory mechanism . Root exposure to ammonium decreased both, cortical cell length and meristem size in a dose-dependent manner and to a similar extent (Fig S1H -J). Since cytokinins determine root meristem size by controlling cell differentiation [START_REF] Dello Ioio | Cytokinins determine Arabidopsis rootmeristem size by controlling cell differentiation[END_REF]), J o u r n a l P r e -p r o o f we checked the response of cytokinin-sensitive reporter line TCS:GFP in the primary root apex, which showed highest expression in the columella and lateral root cap and slight but significant repression under ammonium, revealing that cytokinin signaling was not enhanced by ammonium [START_REF] Bielach | Spatiotemporal regulation of lateral root organogenesis in Arabidopsis by cytokinin[END_REF]Fig S2A, B). The histidine kinase AHK3 acts as a cytokinin receptor to stimulate a two-component signaling pathway that transfers a phosphorelay signal to the nucleus, where transcription factors of the type-B ARABIDOPSIS RESPONSE REGULATOR gene family (ARR-B) are activated [START_REF] Ferreira | Cytokinin signaling[END_REF]. We therefore examined ammonium sensitivity via measuring primary root length and meristem size in the ahk3-3, arr1-3 and arr12-1 mutants, which however revealed no difference to the wild-type (Fig S2C-F), indicating that the AHK3/ARR1 and AHK3/ARR12 two-component cytokinin signaling pathway was not targeted by ammonium. SHY/IAA3 acts directly downstream of ARR1 and ARR12, which increases cell differentiation rate and balances root-meristem size at the transition zone [START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF]. However, neither the SHY2 deletion line shy2-31 nor shy2-2, which expresses a stabilized proteoform of SHY2 [START_REF] Tian | Arabidopsis SHY2/IAA3 inhibits auxinregulated gene expression[END_REF], exhibited altered ammonium sensitivity of primary root elongation or meristem formation (Fig S2G -J). Taken together, these observations indicated that cytokinin signaling is not involved in ammoniumdependent inhibition of root elongation.

As ammonium has been reported to enhance ROS levels in plants [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF][START_REF] Xie | Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa[END_REF], we stained H2O2 in primary roots by 3,3'-diaminobenzidine (DAB). Compared with nitrate, increasing ammonium supply gradually enhanced repression of cell length and meristem size with the detection of H2O2 particularly in the elongation and differentiation zones of the root apex (Fig 1A -G). Since nitratesupplied plants also showed similar DAB staining as ammonium-exposed plants in the meristematic root zone, this zone was not considered as a primary target of ammonium-induced ROS production. To better understand the source of ammoniumdependent ROS formation in primary roots, we employed a pharmacological approach and first applied potassium iodide (KI), acting as a chemical H2O2 scavenger [START_REF] Lee | A mechanism for localized lignin deposition in the endodermis[END_REF]. KI largely reverted the ammonium-dependent inhibition of the primary root elongation rate and restored cell length and meristem size to a large extent (Fig 1A -F). Recovered root elongation coincided with lower abundance of DAB-stained ROS in the elongation and differentiation zone of the primary root J o u r n a l P r e -p r o o f (Fig 1G), suggesting that enhanced levels of H2O2 are responsible for the stunted root phenotype. When ammonium-grown roots were exposed to elevated H2O2 levels, which were generated either directly by supply of 1 mM H2O2 or indirectly by supply of the peroxidase inhibitor salicylhydroxamic acid (SHAM), primary root elongation rates declined drastically due to strongly decreased cell length and meristem size, going along with enhanced DAB staining (Fig S3A -G). Notably, in nitrate-supplied roots, meristem size and DAB staining were apparently not affected by H2O2 or SHAM, while cortical cell length was only slightly decreased (Fig S3D -G), relating to the fact that nitrate-supplied plants possess elevated ROS scavenging capacities [START_REF] Chu | HBI transcription factor-mediated ROS homeostasis regulates nitrate signal transduction[END_REF]. To distinguish between the contribution of superoxide radicals (O2˙-) and H2O2, we investigated the effects of sodium diethyldithiocarbamate (DDC), an inhibitor of Cu,Zn-superoxide dismutase (SOD) that blocks H2O2 generation and leads to O2˙-accumulation [START_REF] Auh | Plasma-membrane redox enzyme is involved in the synthesis of O2and H2O2 by phytophthora elicitor-stimulated rose cells[END_REF]. Both cell length and meristem size of the primary root apex were partially but significantly rescued by DDC supplementation, allowing partial recovery of primary root elongation (Fig S4). This observation indicated that O2˙-radicals are not the direct cause for ammoniummediated inhibition of root elongation, whereas SOD-catalyzed conversion of O2˙-to H2O2 matters. Alternatively, we suspected RBOH-type NADPH oxidases produce ammonium-dependent ROS and examined a couple of rboh single or multiple knockout lines. However, none of them showed an ammonium-dependent root phenotype (Fig S5).

Since in the absence of ammonium H2O2 was less effective in inhibiting root elongation (Fig S3), we assumed ammonium-facilitated formation of H2O2 plays a role and speculated that this process depends on the availability of Fe [START_REF] Dixon | The role of iron and reactive oxygen species in cell death[END_REF]. Indeed, lowering Fe supply from 100 µM to 10 µM Fe(III)-EDTA resulted in weaker inhibition of cell length, meristem size and primary root elongation [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF].

While nitrate-grown roots accumulated some Fe in the meristematic zone, ammonium-exposed roots showed much higher Fe accumulation especially in the differentiation zone (Fig 2D). Cross sections of Perls/DAB-stained primary roots showed that ammonium-dependent Fe precipitation was restricted by the Casparian band and thus confined to the apoplast of outer root cells (Fig S6). Since Fe availability increases with decreasing pH and ammonium nutrition is known to acidify the rhizosphere and apoplast (Römheld and Marschner, 1986;[START_REF] Meier | Auxinmediated root branching is determined by the form of available nitrogen[END_REF], apoplastic pH changes were traced by using the apo-pHusion line, a ratiometric reporter of apoplastic pH [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF]. Increasing ammonium nutrition decreased apoplastic pH in particular in the elongation and differentiation zone of To verify an impact of ROS on cell division, we evaluated the response to ammonium of the cell cycle reporter CycB1;1::GUS (Colón-Carmona et al., 1999). Ammonium supply significantly suppressed GUS activity in the apical meristem of primary root, indicating that oxidative stress acts as a common inhibitory mechanism underlying ammonium-induced growth repression in both primary and lateral roots.

To manipulate Fe availability in the presence of ammonium, medium pH was buffered at different values (Fig S10). While ammonium-inhibited root elongation was aggravated when roots were shifted from standard pH 5.7 to pH 5.0, higher pH restored root elongation leading to almost similar length of nitrate-and ammoniumgrown roots at pH 7.2 (Fig 3A -C). After 6 d of ammonium exposure, Perls/DAB staining revealed decreasing Fe accumulation in the apical root zone with increasing pH, except for the columella cells (Fig 3D). In contrast, strongly enhanced amounts of Fe accumulated at pH 5.0 but only in ammonium-exposed roots. Since the pH of the agar medium was buffered by MES, we suspected that buffer strength might affect ammonium-induced pH changes and Fe availability under ammonium supply. Indeed, lowering MES concentration from 2.5 to 0.5 mM dramatically aggravated Fe precipitation and inhibition of ammonium-exposed primary root tips, while at 10 mM MES Fe precipitation decreased and ammonium-repressed root elongation was partially attenuated (Fig S11). These results indicated that the intensity of ammonium-dependent proton release and acidification of the rhizosphere is of immediate relevance for Fe mobilization and precipitation in the root apoplast and at the root surface.

The dependence of ammonium-induced root growth inhibition on Fe recalls the role of Fe in root growth inhibition under phosphate deficiency that also relies on Fefacilitated ROS formation (Müller et al., 2015). Recently, it has been reported that this role of Fe represents a side effect of light exposure when roots are grown in transparent Petri dishes [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF]. Thus, we shaded the whole root system by aluminium foil as described in [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF] and noted that in all N treatments primary root length increased when roots were shaded from white light (Fig S12A ,B). Even though ammonium-dependent primary root growth inhibition was slightly alleviated by shielding roots from light, ammonium supply still arrested primary root elongation to a large extent (Fig S12B ,C). This observation supported that light increases the pool of redox-active Fe [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF], which in the presence of ammonium may engage in ROS formation and inhibit primary root elongation.

J o u r n a l P r e -p r o o f

Previous work under phosphate deficiency showed that blue light exposure promotes the Fenton reaction converting H2O2 to hydroxyl radicals that are more toxic oxygen species and strongly suppress primary root elongation [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF]. Here, to evaluate the involvement of hydroxyl radicals in ammoniumdependent root growth inhibition, we supplied thiourea, a chemical scavenger of hydroxyl radicals [START_REF] Wasil | The specificity of thiourea, dimethylthiourea and dimethyl sulphoxide as scavengers of hydroxyl radicals. Their protection of alpha 1antiproteinase against inactivation by hypochlorous acid[END_REF], together with ammonium to the medium.

However, the presence of thiourea hardly altered the sensitivity of primary roots to ammonium (Fig S13), indicating that the formation of hydroxyl radicals via the Fenton reaction is not critical for ammonium-dependent root growth inhibition.

Hypersensitivity of root elongation to ammonium relates to vitamin B6 deficiency

To explore how plants cope with ammonium-induced H2O2 formation and subsequent root growth inhibition, we searched in publicly available transcriptome data for ammonium-responsive genes that are expressed in roots [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF][START_REF] Ristova | Combinatorial interaction network of transcriptomic and phenotypic responses to nitrogen and hormones in the Arabidopsis thaliana root[END_REF]. We then collected T-DNA insertion lines of 29 ammoniumresponsive genes and screened them for primary root length under ammonium versus nitrate supply (Data Set S1). In this screen, we identified a T-DNA insertion line of PDX1.1 (SALK_024245), which was hypersensitive to ammonium treatment (Fig S14A ,B). In Arabidopsis, PDX1.1 (along with PDX2) catalyzes vitamin B6 biosynthesis de novo [START_REF] Tambasco-Studart | Vitamin B6 biosynthesis in higher plants[END_REF]Fig S15), thus pdx1.1 mutants suffer from vitamin B6 deficiency [START_REF] Titiz | PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis[END_REF][START_REF] Wagner | Analysis of the Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis[END_REF][START_REF] Boycheva | Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis[END_REF]. We confirmed the hypersensitive phenotype of pdx1.1 mutants to ammonium by examining primary root growth in SALK_024245 and the transposon insertion line pdx1.1-1 (Figure S14C-E). Since SALK_024245 and pdx1.1-1 showed the same growth phenotype under ammonium supply in all our experiments, we focused in the following on SALK_024245, naming it pdx1.1-3. Given that under nitrate supply primary root growth of pdx1.1-3 was weaker (

J o u r n a l P r e -p r o o f

To confirm whether the hypersensitivity of pdx1.1-3 to ammonium is due to the defect in vitamin B6 biosynthesis, we supplied vitamin B6 in the form of pyridoxine to the growth medium. While external supply of pyridoxine to nitrate-grown wild-type plants had no effect, it largely alleviated ammonium-induced inhibition of primary root growth in the wild type and particularly in the pdx1.

Non-phosphorylated forms of vitamin B6 are critical for ammonium detoxification

Vitamin B6 is essential for all living organisms, and refers to a group of six different vitamers that contain a pyridine ring and include the non-phosphorylated forms pyridoxal (PL), pyridoxine (PN) and pyridoxamine (PM), as well as the phosphorylated forms pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) [START_REF] Fitzpatrick | Vitamin B6 in plants: more than meets the eye[END_REF]Fig S15). To evaluate their efficacy in mitigating root growth inhibition, we supplied different B6 vitamers to the medium. Compared with the mock treatment, external supply of PLP slightly retarded the ceasing primary root elongation during the first 4 d after transfer to ammonium, but it hardly restored primary root length after 6 d of ammonium supply (Fig S18A -C).

By contrast, exogenous application of the vitamin B6 forms PL or PN effectively rescued primary root growth under ammonium supply, even during later stages of the treatment, indicating a superior efficacy of the non-phosphorylated B6 vitamers.

When monitoring the H2O2 status in root tips in parallel with root elongation, exogenous application of PN or PL but not of PLP quenched excess accumulation of H2O2 in ammonium-treated primary root tips (Fig S18D). These results reveal that mitigation of root growth inhibition refers to the non-phosphorylated forms of vitamin B6 and their ability to suppress H2O2 accumulation.

J o u r n a l P r e -p r o o f

Over-expression of PDX1.1 improves ammonium tolerance in roots

To address the question whether PDX1.1 plays a role in eliminating ammoniumtriggered ROS, we inspected three independent PDX1.1 overexpression lines [START_REF] Raschke | Enhanced levels of vitamin B-6 increase aerial organ size and positively affect stress tolerance in Arabidopsis[END_REF]. All three lines continued elongating their primary roots even Surprisingly, the concentration of PLP was not enhanced in ammonium-supplied PDX1.1 overexpression lines, although PLP contributed > 70% to overall vitamin B6 levels. In contrast, PDX1.1 overexpression lines showed significantly higher concentrations of non-phosphorylated vitamin B6 forms than the wild-type, and these J o u r n a l P r e -p r o o f differences closely reflected the relative differences in transcript levels among the three overexpression lines. Hence, the proportion of individual B6 vitamers shifted towards the non-phosphorylated vitamin B6 forms, among which PL was the most abundant form contributing up to 25% of total vitamin B6 (Fig 6B). PM accounted only for approx. 1% of total vitamin B6 in roots. In wild-type roots, 20% of total vitamin B6 was in non-phosphorylated forms, while in pdx1.1-3 this proportion was less than 10%. In PDX1.1 overexpression lines, between 30% and 40% of total vitamin B6 was converted into non-phosphorylated forms and this share even increased when plants were grown in the presence of ammonium (Fig 6A ,B). We finally correlated the levels of individual B6 vitamers with those of PDX1.1 transcripts and revealed close correlations for PN, PL, and PM, but not for PMP, whereas the correlation for PLP was also significant although much weaker (Fig 6D). These results indicate that the extent of PDX1.1 upregulation by ammonium or by ectopic expression determines primarily the abundance of non-phosphorylated B6 vitamers without compromising homeostasis of phosphorylated B6 vitamers in roots.

H2O2 triggers localized upregulation of PDX1.1 under ammonium supply

To investigate the transcriptional regulation of vitamin B6 biosynthesis and homeostasis by ammonium, the transcript levels of all known genes involved in vitamin B6 biosynthesis or metabolism were examined by quantitative real-time PCR in roots grown under different N supply. Unexpectedly, PDX1.1 turned out to be the sole vitamin B6-related gene that was significantly upregulated by ammonium (Fig 7A). Next, the tissue-specific expression pattern of PDX1.1 was monitored in plants expressing a PDX1.1 promoter-GUS reporter construct [START_REF] Boycheva | Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis[END_REF]. Given that the change of pH upon ammonium exposure is the initial event to trigger the Fe-dependent oxidative burst, we hypothesized that the transcriptional response of PDX1.1 to ammonium should be influenced by medium pH. Indeed, when pPDX1.1:GUS reporter lines were shifted from pH 5.7 to 5.0, the upregulation of PDX1.1 was enhanced in the presence of ammonium. By contrast, when raising medium pH from 5.7 to 6.5 or 7.2, the induction of PDX1.1 by ammonium almost disappeared (Fig S20). These findings confirmed that ammonium-and pH-dependent H2O2 generation is required to induce the expression of PDX1.1 in roots.

Elevation of PDX1.1-mediated vitamin B6 biosynthesis improves tolerance to further nutrient-related stresses

Apart from ammonium toxicity, other nutrient-related stresses also arrest root elongation by elevated ROS formation, such as phosphate deficiency or nickel toxicity (Müller et al., 2015;[START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF]Lesková et al., 2020). We wondered whether PDX1.1-mediated vitamin B6 biosynthesis also counteracts these oxidative stresses in plants. First, we inspected published transcriptome results [START_REF] Bhosale | A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate[END_REF]Lesková et al., 2020), to assess the response of vitamin B6-related genes to phosphate deficiency and nickel toxicity. Surprisingly, none of the known genes involved in vitamin B6 biosynthesis or metabolism, including PDX1.1, was differentially expressed under phosphate deficiency or nickel toxicity (Fig S21A ,B).

Next, the arrest of primary root elongation under phosphate deficiency or nickel toxicity showed no difference between wild-type (Col-0) and pdx1.1-3 mutant plants (Fig S21C -F). These results suggested that vitamin B6-dependent ROS protection did not evolve as a strategy to alleviate oxidative stress in response to phosphate deficiency or nickel toxicity in natural conditions. Nonetheless, coinciding with their enhanced ROS scavenging capacity, all three PDX1.1 overexpression lines exhibited significantly improved root elongation under phosphate deficiency or nickel toxicity (Fig S21C -F). This result suggests that enhanced PDX1.1-mediated vitamin B6 J o u r n a l P r e -p r o o f biosynthesis can be applied as a practical strategy to improve the root tolerance to multiple types of oxidative stress.

DISCUSSION

Application of ammonium-based N fertilizers in agricultural plant production bears the risk of impaired root development when roots are exposed to ammonium-rich soil patches [START_REF] Britto | NH4 + toxicity in higher plants: a critical review[END_REF][START_REF] Watt | Rates of root and organism growth, soil conditions, and temporal and spatial development of the rhizosphere[END_REF]. Plants have evolved several strategies to cope with the adverse effects of predominant ammonium nutrition, which comprise enhanced N assimilation in roots (Cruz et al., 2005;[START_REF] Guan | Cytosolic glutamine synthetase Gln1;2 is the main isozyme contributing to GS1 activity and can be up-regulated to relieve ammonium toxicity[END_REF][START_REF] Konishi | Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots[END_REF], ammonium compartmentalization to the apoplast or vacuole (Loqué et al., 2005;Li et al., 2010;[START_REF] Bai | A receptor-like kinase mediates ammonium homeostasis and is important for the polar growth of root hairs in Arabidopsis[END_REF], and activation of enzymatic antioxidation systems to cope with ammonium-triggered ROS production [START_REF] Patterson | Distinct signalling pathways and transcriptome response signatures differentiate ammonium-and nitrate-supplied plants[END_REF][START_REF] Xie | Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa[END_REF]. In this study, we describe the mechanistic basis underlying ammonium-triggered ROS formation and identify with PDX1.1-dependent vitamin B6 synthesis a metabolic defense response to counteract ammonium-induced ROS formation.

Ammonium-dependent acidification triggers ROS formation via enhanced Fe mobilization

Ammonium toxicity belongs to one of several mineral element disorders that associate root growth inhibition with the production of ROS (Fig 1; [START_REF] Xie | Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa[END_REF].

Among those disorders, localization and generation of ROS differ in an elementspecific manner, pointing to different sources of ROS formation. For instance, root growth inhibition by nickel has been related to ROS formation in outer cells of the apical root zone (Leskova et al., 2020), while ROS accumulate predominantly in the meristem and elongation zone under zinc deficiency [START_REF] Nakayama | Manganese treatment alleviates zinc deficiency symptoms in Arabidopsis seedlings[END_REF] or in the root vasculature from the meristematic through the elongation and differentiation zone of salt-stressed plants [START_REF] Jiang | ROS-mediated vascular homeostatic control of root-to-shoot soil Na delivery in Arabidopsis[END_REF]. Considering that experimental visualization and localization of ROS depend not only on the site of generation but also on chemical properties of the ROS-sensitive dye or reporter, we used here DAB On the other hand, high effectivity of SOD inhibition by DDC implied that DAB-stained H2O2 was generated via O2˙-, which in turn may derive from peroxisomes and the mitochondrial electron transport chain or from the activity of NADPH oxidases in the plasma membrane [START_REF] Smirnoff | Hydrogen peroxide metabolism and functions in plants[END_REF]. By examining a limited number of available rboh single or multiple knock-out lines, we could not find evidence for a role of RBOH-type NADPH oxidases in ammonium-triggered root growth inhibition (Fig S5), even though our assay also included the root stele-localized RBOHF, which triggers vascular ROS formation upon salinity as a prerequisite for salt tolerance [START_REF] Jiang | ROS-mediated vascular homeostatic control of root-to-shoot soil Na delivery in Arabidopsis[END_REF]. Irrespective of the O2˙-source, SOD-mediated dismutation of O2˙to H2O2 consumes protons and is favored by low apoplastic pH [START_REF] Smirnoff | Hydrogen peroxide metabolism and functions in plants[END_REF] that resulted here from ammonium uptake-induced proton secretion (Fig 2 Indeed, exposure of ammonium-grown roots to light, which increases the pool of redox-active Fe [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF], aggravated inhibition of root elongation (Fig S12). Furthermore, redox-active Fe(III) likely arose from apoplastic acidification and dissolution of apoplastic Fe pools (Fig S6;Zhu et al., 2018) as well as from UVdependent photooxidation of EDTA that sets chelated ferric Fe free [START_REF] Hangarter | Effect of Fe-catalyzed photooxidation of EDTA on root-growth in plant culture media[END_REF]. A similarly critical role of ferrous Fe in ROS-mediated root growth inhibition is also known for primary root growth inhibition under phosphate starvation (Müller et al., 2015;[START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF]. In this context, exposure of P-J o u r n a l P r e -p r o o f deficient roots to light favors reduction of ferric to ferrous Fe and subsequent Fe 2+mediated formation of hydroxyl radicals via the Fenton reaction. DAB-stained H2O2 under P deficiency is confined to the meristematic root zone and further depends on malate efflux via ALMT1 [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF] to increase Fe solubility in the apoplast [START_REF] Balzergue | Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation[END_REF]Mora-Macías et al., 2017). Since the root elongation in

Plant roots counteract ammonium-induced ROS formation via PDX1.1mediated vitamin B6 biosynthesis

In plants, vitamin B6 is synthesized via the "deoxyxylulose-5-phosphate (DXP)independent pathway", which utilizes ribose-5-phosphate, glyceraldehyde-3phosphate and glutamine as substrates for the glutamine amidotransferase complex comprised of pyridoxine synthase (PDX1) and pyridoxine glutaminase (PDX2) [START_REF] Tambasco-Studart | Vitamin B6 biosynthesis in higher plants[END_REF]. The primary product is the phosphorylated form PLP [START_REF] Tambasco-Studart | Vitamin B6 biosynthesis in higher plants[END_REF] before enzymes of the salvage pathway facilitate the interconversion among different B6 vitamers [START_REF] Colinas | Balancing of B-6 vitamers is essential for plant development and metabolism in Arabidopsis[END_REF]Fig S15). While PLP acts as coenzyme in numerous enzymatic reactions, including those with importance for N assimilation [START_REF] Percudani | A genomic overview of pyridoxal-phosphatedependent enzymes[END_REF][START_REF] Fitzpatrick | Vitamin B6 in plants: more than meets the eye[END_REF][START_REF] Colinas | Balancing of B-6 vitamers is essential for plant development and metabolism in Arabidopsis[END_REF], non-phosphorylated forms of vitamin B6 serve efficiently as antioxidants in vitro and in vivo [START_REF] Bilski | Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants[END_REF][START_REF] Havaux | Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress[END_REF] that can boost vitamin B6 biosynthesis via heteromerization with its paralogs in response to singlet oxygen or heat stress [START_REF] Moccand | The pseudoenzyme PDX1.2 boosts vitamin B-6 biosynthesis under heat and oxidative stress in Arabidopsis[END_REF]. Although spatial and temporal expression patterns of PDX1.1 and PDX1.3 largely overlap and only disruption of both genes causes embryo lethality, PDX1.3 has been found to be more abundant and requisite for stress tolerance than PDX1.1 [START_REF] Titiz | PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis[END_REF].

Nonetheless, enhanced expression of PDX1.3 at the protein level appears to require PDX1.2 (Dell' Aglio et al., 2017), whereas overexpression of PDX1.1 can be achieved with the protein alone to substantially increase vitamin B6 production [START_REF] Raschke | Enhanced levels of vitamin B-6 increase aerial organ size and positively affect stress tolerance in Arabidopsis[END_REF]Fig 6). This regulatory versatility of PDX1.1 may provide an advantage when plants need to respond instantly to oxidative stresses. singlet molecular oxygen ( 1 O2; [START_REF] Bilski | Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants[END_REF]. Upon quenching of 1 O2 the pyridoxine ring is degraded [START_REF] Bilski | Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants[END_REF], explaining the dose-dependent rather than catalytic effect of vitamin B6 as observed here (Fig 5,6) al., 2005;[START_REF] Fitzpatrick | Two independent routes of de novo vitamin B6 biosynthesis: not that different after all[END_REF][START_REF] Boycheva | Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis[END_REF]. Glutamine is also the most abundant product of ammonium assimilation in roots [START_REF] Xu | Plant nitrogen assimilation and use efficiency[END_REF]Liu and von Wirén, 2017), which is mediated by cytosolic glutamine synthetase. Interestingly, GLN1;2-mediated glutamine synthesis preferentially localizes in the root vasculature and GLN1;2 represents the most strongly upregulated GLN isoform in ammoniumsupplied roots [START_REF] Ishiyama | Kinetic properties and ammonium-dependent regulation J o u r n a l P r e -p r o o f of cytosolic isoenzymes of glutamine synthetase in Arabidopsis[END_REF][START_REF] Lothier | The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting[END_REF][START_REF] Guan | Cytosolic glutamine synthetase Gln1;2 is the main isozyme contributing to GS1 activity and can be up-regulated to relieve ammonium toxicity[END_REF].

Alternatively, ammonium may also be used directly by PDX1.1, independently of PDX2 action, as has been demonstrated in vitro [START_REF] Raschle | Reaction mechanism of pyridoxal 5 '-phosphate synthase -Detection of an enzyme-bound chromophoric intermediate[END_REF]. In this context, elevated availability not only of glutamine but also of ammonium as one educt for vitamin B6 synthesis in those cells that suffer most from ROS production may be a pathway bringing vitamin B6 from the vasculature to the outer root cells, driven either by diffusion of non-phosphorylated B6 vitamers that are considered able to permeate membranes [START_REF] Stolz | Tpn1p, the plasma membrane vitamin B6 transporter of Saccharomyces cerevisiae[END_REF], or by radial transport via membrane proteins, such as purine permeases (PUPs). In particular PUP1 has been shown to transport non-phosphorylated B6 vitamers in Arabidopsis as well as after heterologous expression in yeast [START_REF] Szydlowski | Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis[END_REF].

Vitamin B6 has proven effective in ROS detoxification in a variety of systems and conditions. In different human cell cultures, the supplementation of nonphosphorylated forms of vitamin B6 alleviates superoxide-induced damage and lipid peroxidation [START_REF] Jain | Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na + +K + )-ATPase activity reduction in high glucose-treated human erythrocytes[END_REF][START_REF] Kannan | Effect of vitamin B-6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H2O2-treated U937 monocytes[END_REF][START_REF] Mahfouz | Vitamin B6 compounds are capable of reducing the superoxide radical and lipid peroxide levels induced by H2O2 in vascular endothelial cells in culture[END_REF]. In Arabidopsis protoplasts, PN supplementation can reduce oxidative damage generated upon high illumination [START_REF] Danon | Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana[END_REF]. When leaf discs were exposed directly to 1 O2, lipid peroxidation in the pdx1.3 background was higher than in the wild type [START_REF] Havaux | Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress[END_REF]. Since this effect was not observed with O2˙-or H2O2, and since 1 O2 levels rise during illumination more drastically in the pdx1.3 mutant than in the wild type, vitamin B6 has been proposed to act as 1 O2 quencher [START_REF] Danon | Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana[END_REF][START_REF] Havaux | Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress[END_REF]. Moreover, 1 O2 can easily convert to O2˙-by electron transfer [START_REF] Khan | Singlet molecular-oxygen in the Haber-Weiss reaction[END_REF] and is further converted to H2O2 by SOD, allowing reactive Fe to take in a key role in ammonium-induced ROS processing (Fig 7D). Our study could not identify the source of produced 1 O2 or O2˙-species, as analysis of a few tested rboh mutants did not provide sufficient evidence to rule out NADPH oxidases as a source for O2˙-formation (Fig S5). Alternatively, 1 O2 or O2˙-species may derive from electron transport processes in root plastids or mitochondria [START_REF] Smirnoff | Hydrogen peroxide metabolism and functions in plants[END_REF]. Irrespective of their origin, their accumulation during repression of SOD by DDC was ineffective in inhibiting root elongation (Fig S4 ), indicating that the conversion of O2˙-to H2O2 by SOD was required to inhibit root elongation (Fig 7D). Actually, these two ROS species greatly influence root development by regulating the balance between cell proliferation and cell differentiation in the root tips [START_REF] Tsukagoshi | Control of root growth and development by reactive oxygen species[END_REF]. Specifically, O2˙-localized in the meristematic zone maintains cell division, while H2O2 prevailing in the elongation zone stimulates cell differentiation [START_REF] Tsukagoshi | Transcriptional Regulation of ROS Controls Transition from Proliferation to Differentiation in the Root[END_REF]. However, excessive accumulation of H2O2 in the elongation zone leads to a repression of root growth reflected by inhibited cell elongation and smaller meristem size (Tsukagoshi et Based on our study, we propose a working model of the processes underlying primary root growth inhibition under ammonium nutrition (Fig 7D). Ammonium uptake, which is particularly high in the elongation zone [START_REF] Duan | Root zonespecific localization of AMTs determines ammonium transport pathways and nitrogen allocation to shoots[END_REF], provokes apoplastic acidification [START_REF] Meier | Auxinmediated root branching is determined by the form of available nitrogen[END_REF] formation to restore root growth. We find that this PDX1.1-mediated biosynthesis de novo of vitamin B6 is not only essential for protecting roots against ammonium toxicity, as occurring in ammonium-enriched fertilizer bands in agricultural plant production, but also effective against other adverse growth conditions that involve Fe-dependent ROS formation such as P deficiency and nickel toxicity.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana accession Col-0 and Ler served as wild type. The following mutants and transgenic lines were used: ahk3-3 (Dello loio et al., 2007), arr1-3 (Dello loio et al., 2007), arr12-1 (Dello loio et al., 2007), shy2-31 (Dello loio et al., 2008), shy2-2 (Dello loio et al., 2008), TCS:GFP [START_REF] Bielach | Spatiotemporal regulation of lateral root organogenesis in Arabidopsis by cytokinin[END_REF], pdx1.1-1 [START_REF] Titiz | PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis[END_REF], pdx1.1-3 (SALK_024245), apo-pHusion [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF], PDX1.1OE-L5 [START_REF] Raschke | Enhanced levels of vitamin B-6 increase aerial organ size and positively affect stress tolerance in Arabidopsis[END_REF], PDX1.1OE-L8 [START_REF] Raschke | Enhanced levels of vitamin B-6 increase aerial organ size and positively affect stress tolerance in Arabidopsis[END_REF], PDX1.1OE-L15 [START_REF] Raschke | Enhanced levels of vitamin B-6 increase aerial organ size and positively affect stress tolerance in Arabidopsis[END_REF], pPDX1.1:GUS [START_REF] Boycheva | Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis[END_REF], CycB1;1::GUS or 5 µM KH2PO4 (-P), KCl was added to -P medium to balance K + concentrations. For Ni toxicity, 6 days-old plants were transferred to half-strength MS medium in the absence (-Ni) or presence (+Ni) of 75 µM NiSO4. Root phenotypes were assessed 6 d after transfer. When supplementing H2O2, fresh 30% H2O2 (Roth) solution (9.79 mol L -1 ) was diluted to a concentration of 1 M and supplied to the medium after autoclaving. In the root shading experiment, root-containing segments of Petri dishes were covered with aluminium foil as described in [START_REF] Zheng | Blue light-triggered chemical reactions underlie phosphate deficiency-induced inhibition of root elongation of Arabidopsis seedlings grown in Petri dishes[END_REF]. For vitamin B6 supplementation experiments, different B6 vitamers were dissolved in water and supplied to agar medium at a final concentration of 5 µM. Key chemical information is given in Table S1.

Root growth measurements

During root phenotyping experiments, the position of primary roots was labeled every day after treatment in order to calculate primary root elongation rate. Root images were taken by an Epson 10000XL scanner at a resolution of 300 dpi. Primary root growth parameters were measured by WinRHIZO Pro 2007 software (Regents Instruments Canada). Differential interference contrast (DIC) microscopy images of primary root tips were taken to assess the size of primary root meristem and the J o u r n a l P r e -p r o o f length of mature cortical cell as described in Dello [START_REF] Dello Ioio | Cytokinins determine Arabidopsis rootmeristem size by controlling cell differentiation[END_REF]. All the experiments were performed at least twice with similar results.

ROS detection

H2O2 in primary root tips were detected by 3,3'-Diaminobenzidine (DAB) staining as described by [START_REF] Thordal-Christensen | Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction[END_REF]. Briefly, primary roots were incubated in 1 mg ml -1 DAB solution for 8 h, and then imaged by DIC microscopy. H2O2 is visualized as a reddish-brown coloration. A cell-permeant fluorogenic dye 2',7'dichlorodihydrofluorescein diacetate (H2DCFDA) was also used to measure ROS activity in primary roots. Seedlings were stained for 20 min in a solution of 50 µM H2DCFDA in 50 mM potassium phosphate buffers (pH 7.0). DCF fluorescence was excited at 488 nm and detected at 517-527 nm. Virtual color images were generated by a rainbow color code to indicate the fluorescence intensity of DCF in roots.

Apoplastic and rhizosphere pH measurements

Apoplastic pH changes were measured by the ratiometric pH reporter apo-pHusion [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF]. The fluorescence intensity ratio between pH-sensitive GFP and pH-insensitive RFP was calculated to indicate the changes of apoplastic pH, and virtual ratio images were generated by ImageJ v1.53 software. The changes of rhizosphere pH under different N supply were monitored by the pH indicator bromocresol purple (BCP) that changes color from yellow at pH 5.0 to violet at pH 7.2 [START_REF] Meier | Auxinmediated root branching is determined by the form of available nitrogen[END_REF]. Six days after growth on different N sources, agar medium containing six seedlings per plate were stained overnight (18 h) with BCP solution at a final concentration of 0.1 mg ml -1 .

Histological staining

Promoter-driven β-glucuronidase (GUS) activity was determined by GUS staining as described previously [START_REF] Li | Histostaining for tissue expression pattern of promoter-driven GUS activity in Arabidopsis[END_REF]. Roots were rinsed once with staining buffer containing 50 mM NaHPO4 buffer (pH 7.2), 2 mM potassium ferrocyanide, and 2 mM potassium ferricyanide, and then incubated at 37°C in staining solution containing 2 mM X-Gluc. After staining, roots were cleared by HCG solution (chloral hydrate:water:glycerol = 8:3:1) and imaged by DIC microscopy. To assess the size of the primary root meristem and the length of mature cortical cell, non-staining primary root tips were cleared by HCG solution and imaged by DIC microscopy. To stain Fe accumulation in roots, a Perls staining and DAB/H2O2 intensification was performed according to [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF]. Roots were rinsed three times with 10 mM EDTA, and then incubated for 5 min in a freshly prepared Perls staining solution.

J o u r n a l P r e -p r o o f

Afterward, roots were incubated in a methanol solution containing 0.01 M NaN3 and 0.3% (v/v) H2O2 for 1 h at room temperature. After washing three times with 0.1 M phosphate buffer (pH 7.4), roots were finally incubated for 5 min in an intensification solution containing 0.025% (w/v) DAB and 0.005% (v/v) H2O2 in 0.1 M phosphate buffer (pH 7.4). Roots were mounted with HCG solution before imaging by light microscopy.

Microscopy analyses

Fluorescent images were taken by laser scanning confocal microscopy Zeiss LSM 780. Root samples were stained with 10 µg ml -1 propidium iodide (PI) for 5 min to visualize cell walls. GFP was excited at 488 nm and detected at 505-535 nm; RFP was excited at 561 nm and detected at 580-630 nm; PI was excited at 561 nm and detected at 600-700 nm. DCF fluorescence was excited at 488 nm and detected at 517-527 nm. The same microscope settings were kept to measure all confocal sections across samples. Fluorescence quantification of apo-pHusion and H2DCFDA were conducted by Zeiss ZEN microscope software (version 2.6). Virtual ratio images were generated by Image J 1.53 software. DAB staining, Perls staining, GUS staining and DIC images were taken by Zeiss Axio Imager 2 system. For light microscopy of in situ-localized Fe, root cuttings of 5 mm length of 5 seedlings from each growth condition, were dissected approximately 1 cm above the root tip and subjected to aldehyde fixation, dehydration and resin embedding as described in Table S2. Semithin sections of 2.5 µm thickness were cut with a Leica UCT microtome (Leica Microsystems, Wetzlar, Germany), and mounted on slides in rapid mounting medium Entelan (Sigma-Aldrich, Darmstadt, Germany). Sections were recorded with a 40x lense at fixed exposure time using a Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

Vitamin B6 quantification analysis

The abundance of all individual B6 vitamers in roots were determined by HPLC.

Vitamin B6 quantification analyses were performed as described previously [START_REF] Colinas | Balancing of B-6 vitamers is essential for plant development and metabolism in Arabidopsis[END_REF] with the following changes: two separate extractions were performed with 15 volumes and 8 volumes of 50 mM ammonium acetate (pH 4.0), respectively, and a 50 µL injection volume was used for a single run per extract.

Real-time quantitative PCR

J o u r n a l P r e -p r o o f

Total RNA was extracted from 10-20 mg frozen root samples by RNeasy plant mini kit (Qiagen) following the manufacturer's protocol. Template cDNA was synthesized from 1 µg total RNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and Oligo d(T)12-18 primer. Real-time qPCR was performed by CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad). According to the multiple internal control method [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF], relative transcript levels of target genes were calculated by the geNorm algorithm (https://genorm.cmgg.be), using UBQ10 (AT4G05320) and ACTIN2 (AT3G18780) as the multiple internal control genes in this study. Gene specific primers for qPCR are listed in Table S3.

Statistical analysis

Data

Accession numbers

Sequence data in this study can be found in the Arabidopsis Information Resource (A) Visual appearance of seedlings subjected to different N supplies. Wild-type plants were pre-cultured on half-strength MS medium containing 1 mM NO3 -for 6 d before being transferred to 1 mM NO3 -, 1 mM NH4 + or 10 mM NH4 + as sole N source. During plant growth, the positions of primary root tips were labeled every day to calculate primary root elongation rates. Images were taken 6 d after transfer. Scale bar = 1 cm. Different letters represent significant differences between treatments and lines according to two-way ANOVA followed by Tukey's HSD test at P<0.05. Different letters represent significant differences between samples according to twoway ANOVA followed by Tukey's HSD test at P<0.05. Gln, glutamine; Glu, glutamate; R5P, ribose 5-phosphate; G3P, glyceraldehyde 3phosphate; PNP, pyridoxine 5'-phosphate; PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phosphate; PN, pyridoxine; PL, pyridoxal; PM, pyridoxamine; P-ase, phosphatase.

J o u r n a l P r e -p r o o f Table S1. Chemical Information. Table S2. Protocol for fixation, dehydration and embedding of root tissue. Table S3. Primers used in this study. Data Set S1. Information on ammonium-responsive genes used for the mutant screening in this study.

J o u r n a l P r e -p r o o f

  and prevented accumulation of DAB-stained ROS in the elongation and differentiation zone of the primary root (Fig 1A-G). Higher resolution of DAB-stained cells allowed assigning H2O2 to the stele of the late elongation and early differentiation zone, which became shorter under increasing ammonium supply and thus appeared more apical (Fig 2A). In addition, we localized ROS-dependent fluorescence by the cell-permeant indicator 2',7'-dichlorofluorescin diacetate (H2DCFDA), revealing enhanced ROS formation in the stele, especially along the vascular strands of ammonium-exposed roots with a maximum at the transition of the J o u r n a l P r e -p r o o f elongation to the differentiation zone (Fig 2B, C). Considering Fe dependency of ammonium-triggered root inhibition (Fig 1), Fe distribution in the root was stained by DAB-enhanced Perls detecting both free Fe(II) and Fe(III)

  ammonium-exposed roots (Fig 2E, F), which co-localized with DAB-or fluorescencestained ROS (Fig 2A-D), suggesting that the ammonium-dependent pH decrease and enhanced Fe availability caused ROS formation. Since ammonium-induced repression of root elongation appears to be a rapid response detected even 1 d after ammonium exposure (Fig S1E-G), we investigated dynamic changes in Fe availability and ROS status in ammonium-supplied primary roots. Enhanced Fe precipitation and ROS accumulation appeared as early as 1 d after ammonium supply in the differentiation zone of the primary root and progressed in the apical direction with time and in dependence of external ammonium supply (Fig S7). As indicated by the arrows marking the boundaries of the meristematic and elongation zones, the size of the elongation zone started declining earlier than that of the meristematic zone, which went along with progression of H2DCFDA-dependent fluorescence into the elongation zone towards the meristematic zone (Fig 2B; S7B).

  and this suppression was largely alleviated by lowering external Fe supply (Fig S8). Consistent with the decrease of meristem size and cell length under the same conditions (Fig 1D-F), we conclude that Fe-dependent ROS formation is a major repressor of both cell elongation and cell division in ammonium-exposed root apices. Notably, exactly the same responses of apoplastic pH, Fe availability and ROS J o u r n a l P r e -p r o o f formation as in primary root were detected in the apex of lateral roots (Fig S9),

  Fig 4A), we calculated the relative primary root elongation rate by normalizing it to the root growth rate under nitrate. An earlier and steeper decrease in root elongation rate of pdx1.1-3 than of the wild type confirmed hypersensitivity of pdx1.1-3 to ammonium nutrition (Fig 4B, C). The higher sensitivity of pdx1.1-3 to ammonium went along with elevated accumulation of H2O2 in ammonium-exposed primary root tips already 2 d after transfer to ammonium (Fig 4D).

  1-3 mutant (Fig 4A). Notably, external supplementation of pyridoxine recovered completely the primary root elongation rate of pdx1.1-3 (Fig 4B-C), cortical cell length and meristem size (Fig S16) as well as GUS activity of the CycB1;1::GUS reporter (Fig S17), confirming that the hypersensitive phenotype of pdx1.1-3 to ammonium is due to defective vitamin B6 biosynthesis. Since external supply of pyridoxine reduced also the accumulation of DAB-stained H2O2 in ammonium-treated primary roots of both lines (Fig 4E), we conclude that vitamin B6 alleviates ammonium toxicity either by suppressing H2O2 generation or by scavenging H2O2 in primary root tips.

  after 6 d of ammonium exposure when root elongation of the wild-type was almost completely exhausted (Fig 5A-C). DAB staining showed that overexpression of PDX1.1 strongly reduced the level of H2O2 in ammonium-treated primary roots (Fig 5D). Accordingly, ammonium-induced inhibition of root cortical cell length, meristem size and activity of the CycB1;1::GUS reporter also recovered by overexpression of PDX1.1 (Fig 5E-G; Fig S19), revealing the efficacy of endogenous vitamin B6 formation under ammonium nutrition. Moreover, an 15 NH4 + influx experiment revealed that the ammonium uptake capacity in roots remained unaffected in the pdx1.1-3 mutant as well as in the PDX1.1 overexpression lines (Fig 5H). Taken together, elevated expression of PDX1.1 is sufficient to restore elongation in ammoniumexposed roots and acts downstream of the ammonium uptake process. Overexpression of PDX1.1 increases the level of non-phosphorylated B6 vitamers As the PDX1-dependent step in vitamin B6 biosynthesis yields first PLP (Fig S15), we hypothesized that overexpression of PDX1.1 will primarily increase phosphorylated rather than non-phosphorylated vitamers, whose generation requires an additional phosphatase reaction. Measuring vitamin B6 in roots confirmed significantly lower levels of total vitamin B6 in the pdx1.1-3 mutant than in the wild type, which was caused by a significant decrease in the concentrations of all five determined B6 vitamers (Fig 6A, B). At first glance, this suggested a constitutive contribution of PDX1.1 to overall vitamin B6 biosynthesis because vitamin B6 levels decreased in pdx1.1-3 irrespective of the supplied N form. Overexpression of PDX1.1 led to constitutively higher total vitamin B6 levels only in PDX1.1OE-L5, but under ammonium supply also in PDX1.1OE-L15 and to lesser extent in PDX1.1OE-L8, which coincided with elevated PDX1.1 transcript levels in these lines (Fig 6A-C).

PDX1. 1

 1 reporter activity was upregulated by ammonium supply in the root vasculature, increasing from the elongation zone basipetally through the differentiation zone (Fig 7B). As the reporter activity became stronger in the presence of 10 mM ammonium, upregulation of PDX1.1 expression apparently followed ammonium supply in a dose-dependent manner (Fig 7A, B). Since the spatial localization of PDX1.1 expression and H2O2 accumulation strongly overlapped in ammonium-treated roots (Fig 2A-B, 7B), the question arose whether ammonium itself or H2O2 triggered the upregulation of PDX1.1. We thus suppressed H2O2 levels in ammonium-exposed roots by supply of KI or by low Fe and observed that PDX1.1 reporter activity disappeared (Fig 7B). By contrast, J o u r n a l P r e -p r o o f exogenous application of 1 mM H2O2 strongly enhanced the promoter activity of PDX1.1 in the root stele as well as in the apical root meristem, however only in the presence of ammonium (Fig 7C). Therefore, H2O2 serves as a signal to upregulate PDX1.1 transcription upon ammonium nutrition and determine its restricted expression in the stele.

  staining and H2DCFDA-dependent fluorescence to localize ROS formation under ammonium nutrition primarily to the vascular cylinder or stele in the elongation and differentiation zones (Fig 2). With increasing ammonium supply and time of exposure ROS formation gradually progressed through the elongation zone towards the J o u r n a l P r e -p r o o f meristem (Fig 2B-C; S7B), where it coincided with suppressed cell length, meristem size and cell division activity (Fig 1; S3; S8), revealing that the apical root meristem is not the primary target of ammonium-dependent ROS generation especially at early stages of ammonium exposure. Although cytokinins are crucial in balancing cell division and meristem size (Dello Ioio et al., 2008), key components of cytokinin signaling are apparently not involved in ammonium-dependent root growth inhibition (Fig S2). Since ammonium-dependent root growth inhibition was suppressed when H2O2 accumulation was chemically quenched by KI (Fig 1) but enhanced when H2O2 accumulation was favored in the presence of SHAM or by H2O2 supplementation (Fig S3), H2O2 or a downstream product must have caused the stunted root phenotype. This is supported by the observation that blocking H2O2 formation by DDC in favor of O2˙-accumulation prevented ammonium-dependent root growth inhibition (Fig S4).

  Fig 7D; Meier et al., 2020). Suppressed DAB staining and mitigation of root growth inhibition under low Fe supply (Fig 1) indicated a key role of Fe in H2O2 formation.

  response to ammonium was not altered in almt1 mutants (Fig S14A, B), root shading restored root elongation only in part (Fig S12) and ammonium-dependent H2O2 mainly localized to the root stele above the meristematic zone (Fig 2), mechanisms underlying ROS formation clearly differ between P-deficient and ammonium-exposed roots. Moreover, supplementation of the hydroxyl radical scavenger thiourea, which can restore arrested primary root elongation under P deficiency (Zheng et al., 2019), was poorly effective in the case of ammonium (Fig S13), suggesting that formation of hydroxyl radicals via the Fenton reaction is not critical for ammonium-dependent root growth inhibition. Nonetheless, a determinant role of redox-active Fe in ammoniumtriggered root growth inhibition is corroborated by its gradual relief under increasing medium pH, which also decreased Fe accumulation in the root and subsequent Fe availability for ROS metabolism (Fig 3). We thus conclude that ammonium-induced acidification of the root apoplast and additionally in the rhizosphere enhance Fe solubilization as prerequisite for re-location to the stele and subsequent Fe-mediated formation of H2O2.

  ; Mooney and J o u r n a l P r e -p r o o f Hellmann, 2010). Our study provides several lines of evidence indicating that plants induce PDX1.1-mediated synthesis of non-phosphorylated B6 vitamers as an efficient strategy to counteract ammonium-dependent oxidative stress in roots. First, as several enzymes are required for vitamin B6 biosynthesis and homeostasis, it was surprising that only one of them, PDX1.1, responded to ammonium with enhanced expression (Fig 7A). Indeed, overexpression of PDX1.1 suppressed ammonium-dependent H2O2 formation and associated root length inhibition, whereas pdx1.1 mutant lines were hypersensitive to ammonium (Fig 4 and 5, Fig S14). As in these lines total vitamin B6 levels closely followed PDX1.1 transcript levels in roots, transcriptional regulation of PDX1.1-dependent PLP synthesis alone provides sufficient plasticity to counteract the adverse growth effect of ammonium. Among the three paralogs of PDX1 in Arabidopsis, only PDX1.1 and PDX1.3 show catalytic activities (Tambasco-Studart et al., 2005), while PDX1.2 encodes a pseudoenzyme

  Second, external supply of vitamin B6 to pdx1.1-3 mutant or wild-type plants completely prevented ammonium-induced inhibition of root elongation (Fig 4). Although the overall vitamin B6 level in the pdx1.1-3 mutant line was only 20-30% lower than in the wild type (Fig 6A), this difference as well as a further 50-100% increase in the overexpressing lines gradually improved root growth, indicating a strong dose dependence of beneficial vitamin B6 action. This dose-dependent effect went back to the abundance of the non-phosphorylated B6 vitamers (Fig 6A), because only PL and PN restored root length while the primary biosynthesis product PLP remained ineffective (Fig S18). Superior functionality of non-phosphorylated vitamers as ROS scavengers is most likely determined by substituents of the pyridoxine core that modulate electron density in the ring and thus the interaction with J o u r n a l P r e -p r o o f

  . Since in overexpression lines root concentrations of the three non-phosphorylated vitamers PM, PN and PL only, correlated closely with PDX1.1 transcript levels (Fig 6D), transcriptional upregulation of PDX1.1 is sufficient to confer a B6 vitamer-specific defense response to stress. Third, PDX1.1-mediated biosynthesis de novo of vitamin B6 spatially and temporally coincides with ammonium-induced ROS formation in roots. Within a time frame of a few days, there was a robust temporal coincidence between root elongation rates and H2O2 accumulation, even when vitamin B6 was provided externally or PDX1.1 expression levels were modulated (Fig 4, 5). Also at the tissue level, spatial patterns of PDX1.1 transcript levels and H2O2 accumulation strongly overlapped in ammonium-treated roots (Fig 2, 7), supporting the notion that vitamin B6 biosynthesis is targeted to those tissues and root developmental zones that are most severely affected by ROS accumulation. In plants, biosynthesis de novo of vitamin B6 relies on PDX2 using glutamine as a substrate to produce PLP (Fig S15; Tambasco-Studart et

  factor why plants favored vitamin B6 as preferential ROS scavenger during evolutionary adaptation to elevated external ammonium levels. Given that ammonium-induced ROS formation occurs in all the cell types of the root differentiation zone and meristem (Fig 2A-C; S7B) while the enhancement of PDX1.1-mediated vitamin B6 biosynthesis under ammonium supply is confined to the vasculature (Fig 7B; S20), the question arises how root cells that do not produce vitamin B6 are protected from oxidative stress. Likely there is a radial transport J o u r n a l P r e -p r o o f

  al., J o u r n a l P r e -p r o o f 2010). This may explain how H2O2 accumulation inhibited root elongation in ammonium-grown plants (Fig 7D).

  that increases Fe solubilization and reprecipitation in inner root cells (Fig 2, 7D). It is not yet completely clear why Fe preferentially accumulates along the stele and whether the required change in Fe binding forms for xylem loading is involved, but previous studies have confirmed enhanced Fe precipitation in the pericycle and xylem (Green and Rogers, 2004; Roschzttardtz et al., 2013). There, elevated Fe availability and acidic pH favor O2˙dismutation and H2O2 formation (Smirnoff an Arnaud, 2018, Fig 7D). Considering that ammonium uptake-dependent acidification stimulates the generation of H2O2, H2O2 or a downstream product induces PDX1.1 expression at the site of Fe and ROS localization (Fig 7B, D). Predominant biosynthesis of non-phosphorylated B6 vitamers (Fig 6) can subsequently quench reactive molecular oxygen ( 1 O2/O2˙-) that serves as source for H2O2 formation (Fig S4) and thereby counteract ammonium-induced H2O2

(

  Colón-Carmona et al., 1999). The cell cycle reporter CycB1;1::GUS (Col-0 J o u r n a l P r e -p r o o f background) was introduced into pdx1.1-3 or PDX1.1OE-L5 by crossing to generate CycB1;1::GUS (pdx1.1-3) and CycB1;1::GUS (PDX1.1OE-L5) lines. Complete information of T-DNA insertion lines of 29 ammonium-responsive genes used in the mutagenesis screen are listed in Data Set S1. Arabidopsis seeds were surface sterilized by 70% ethanol with 0.05% (v/v) Triton X-100, and cultured on modified half-strength Murashige and Skoog (MS) medium containing 100 µM Fe(III)-EDTA, 0.5% sucrose, 1% Duchefa Phyto agar (Duchefa Biochemie), 2.5 mM MES pH 5.7, and N sources were added to different final concentrations as described in the figure legends. Seedlings grown in Petri dishes (12 × 12 cm) were cultured vertically in a growth chamber under a 22°C/18°C and 10/14 h (light/dark) regime at the light intensity of 120 μmol photons m -2 s -1 . For root phenotyping experiments, plants were pre-cultured on half-strength MS medium containing 1 mM KNO3 for 6 d, and then transferred to treatment plates supplemented with half-strength MS medium containing 1 mM KNO3, 1 mM NH4Cl or 10 mM NH4Cl respectively. 0.5 mM K2SO4 was added to balance K + concentration in the ammonium treatment. Root phenotypes were measured 6 d after transfer. To generate P deficiency, 6 days-old plants were transferred to half-strength MS medium containing 625 µM KH2PO4 (+P)

  were collected and analyzed by Microsoft Excel 2016. Statistical analyses were conducted by Graphpad Prism 8 (version 8.3.0). Two-tailed Student's t-test, Dunnett's multiple test or Tukey's HSD test was performed to test the statistical significance, and the P-value of each statistical analysis is described in the figure legends. Graphs were plotted by Graphpad Prism 8 (version 8.3.0), and edited by Adobe Illustrator 2020 (version 24.2.1).

(

  TAIR) according to the following accession numbers: PDX1.1 (AT2G38230), PDX1.2 (AT3G16050), PDX1.3 (AT5G01410), PDX2 (AT5G60540), PDX3 (AT5G49970), SOS4 (AT5G37850), PLR1 (AT5G53580), UBQ10 (AT4G05320) and ACTIN2 (AT3G18780). AUTHOR CONTRIBUTIONS Y.L. and N. v. W. conceived the project and designed the experiments. Y.L., R.F.H.G., R.A.M., M.M., P.S. and T.B.F. performed the experiments and analyzed the data. G.K. provided the transcriptome data. Y.L. and N.v.W. wrote the manuscript with support of R.F.H.G. and T.B.F.

  FIGURE LEGENDS Figure 1. Involvement of H2O2 and Fe in ammonium-dependent root growth inhibition. (A) Root phenotype of wild-type plants 6 d after transfer to different N supply, which contained additionally either 0.5 mM potassium iodide (KI) or 10 µM Fe(III)-EDTA (low Fe) instead of 100 µM Fe(III)-EDTA as in control plates. Horizontal marks along the root axis indicate daily positions of primary root tips. Root images were scanned 6 d after transfer. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) or under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control and indicated treatments at each time point as * P<0.05 ** P<0.01 *** P<0.001 according to Dunnett's multiple test. (D) Cell length and meristem size of primary roots. The length of mature cortical cells and the size of the root apical meristem are indicated by yellow arrowheads. DIC images of primary root tips were taken at 6 d after transfer. Scale bar = 100 µm. (E) -(F) Quantitative readout of mature cortical cell length (E), and primary root meristem size (F). Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 16 plants. Different letters represent significant differences between treatments according to two-way ANOVA followed by Tukey's HSD test, P<0.05. (G) DAB staining of H2O2 in primary root tips at 6 d after treatment. The reddish-brown coloration indicates H2O2. Representative images from 10 plants per treatment are shown. Scale bar = 200 µm.

Figure

  Figure 2. Ammonium-dependent histochemical changes in the primary root apex. (A) DAB staining of H2O2 in primary root tips at 6 d after treatment. The reddishbrown coloration indicates H2O2. Representative images from 10 plants per treatment are shown. Scale bar = 100 µm. (B) Staining of ROS (oxidant levels) by H2DCFDA

Figure 3 .

 3 Figure 3. Influence of medium pH on primary root growth and root tissue Fe. (A) Root phenotype of wild-type plants 6 d after transfer to different N supply buffered at different pH. Medium pH of 5.0, 5.7 and 6.5 was buffered by 2.5 mM MES, while pH 7.2 was buffered by 2.5 mM MOPS. Horizontal marks along the root axis indicate daily positions of primary root tips. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) or under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control (pH 5.7) and indicated treatments at each time point as * P<0.05 ** P<0.01 *** P<0.001 according to Dunnett's multiple test. (D) Perls/DAB staining of Fe as indicated by a reddish-brown color. Perls/DAB staining was conducted at 6 d after transfer. Representative images from 10 plants per treatment are shown. Scale bars: 100 µm.

Figure

  Figure 4. Exogenous application of vitamin B6 alleviates ammonium toxicity in roots. (A) Root phenotype of Col-0 and pdx1.1-3 mutant plants 6 d after transfer to different N supply in the absence or presence of 5 µM vitamin B6 (pyridoxine). Horizontal marks along the root axis indicate daily positions of primary root tips. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) or under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Data represent means ± SE, n = 20 plants per treatment. Different letters represent significant differences at each time point at P<0.05 according to Tukey's HSD test. (D) H2O2 accumulation in the primary root as visualized by DAB staining in wild-type and pdx1.1-3 mutant plants under differing N supply. DAB staining was conducted 2 d after treatment (2DAT) or 6 d after treatment (6DAT). Representative images from 10 seedlings per treatment are shown. Scale bar = 200 µm. (E) DAB staining of H2O2 in primary root tips 6 d after treatment. The reddish-brown coloration indicates H2O2. Representative images from 10 seedlings per treatment are shown. Scale bar = 200 µm.

Figure 5 .

 5 Figure 5. Over-expression of PDX1.1 enhances ammonium tolerance in roots. (A) Root phenotype of wild-type, pdx1.1-3 mutant and three independent PDX1.1 overexpression lines subjected to different N supply. Horizontal marks along the root axis indicate daily positions of primary root tips. After pre-culture of 6 d, plants were transferred to media containing different N forms. Root images were taken at 6 days after transfer. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) or under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between wild type and indicated lines at each time point as * P<0.05 ** P<0.01 *** P<0.001 by Dunnett's multiple test. (D) DAB staining of H2O2 in primary root tips of wild type, pdx1.1-3 and PDX1.1OE-L5 at 6 d after treatment. The reddish-brown coloration indicates H2O2. Representative images from 10 plants per treatment are shown. Scale bar = 200 µm. (E) Cell length and meristem size of primary roots of wild type, pdx1.1-3 and PDX1.1OE-L5. The length of mature cortical cells and the size of the root apical meristem are indicated by yellow arrowheads. DIC images of primary root tips were taken 6 d after treatment. Scale bar = 100 µm. (F) -(G) Quantitative readout of mature cortical cell length (F), and primary root meristem size (G). Boxes show the

Figure 6 .

 6 Figure 6. Vitamin B6 profiling of pdx1.1 mutant and PDX1.1-overexpressing plants under different N regimes. (A) Root concentrations of vitamin B6, pyridoxamine 5'-phosphate (PMP), pyridoxal 5'-phosphate (PLP), pyridoxamine (PM), pyridoxine (PN) and pyridoxal (PL) in wild type (Col-0), pdx1.1-3 mutant and three PDX1.1 overexpression lines 6 d after transfer to different N supply. The amount of pyridoxine 5'-phosphate (PNP) was too low to be detected in this study. Bars represent means ± SD, n = 4 independent biological replicates. Asterisks denote significant differences between wild type and indicated lines at * P<0.05 ** P<0.01 *** P<0.001, by Dunnett's multiple test; ND = not detected. (B) Proportion of individual B6 vitamers in the roots and (C) relative transcript abundance of PDX1.1 in the roots of wild type, pdx1.1-3 and PDX1.1 overexpression lines 6 d after transfer to different N supplies. The relative transcript level of PDX1.1 was determined by quantitative real-time PCR, and normalized by using ACTIN2 and UBQ10 as internal controls. Bars represent means ± SD, n = 4 independent biological replicates. (D) Correlation between the transcript abundance of PDX1.1 and the concentration of individual B6 vitamers in roots of wild type, pdx1.1-3 and PDX1.1 overexpression lines. Pearson's correlation coefficients are shown as R values.

Figure 7 .

 7 Figure 7. Transcriptional regulation of PDX1.1 and other vitamin B6-related genes in response to ammonium. (A) Relative transcript abundance of PDX1.1, PDX1.2, PDX1.3, PDX2, PDX3, SOS4 and PLR1 in roots of wild-type plants 6 d after transfer to different N supplies. Relative transcript levels were determined by quantitative real-time PCR and normalized by using both ACTIN2 and UBQ10 as multiple internal controls. Bars represent means ± SD, n = 3 biological replicates. Different letters represent

(

  B) -(D), Primary root length (B), total lateral root length (C), and mean lateral root length (D) of wild-type plants 6 d after transfer to different N treatments. Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 20 independent plants. Different letters represent significant differences at P<0.05 according to Tukey's HSD test. (E) -(G), Primary root length (E), primary root elongation rate (F), and relative primary root elongation rate (G) of wild-type plants under differing N supplies. Symbols represent means ± SE, n = 20 plants per treatment. Different letters in (E) and (F) represent significant differences within each time point at P<0.05 according to Tukey's HSD test. Different letters in (G) denote significant differences among time points under 1 mM or 10 mM NH4 + respectively, according to Tukey's HSD test. (H) Cell length and meristem size of primary roots. Primary roots were stained by propidium iodide (PI), and confocal images were taken 6 d after transfer. The length of mature cortical cells and the size of the root apical meristem are indicated by red arrowheads. Scale bar = 100 µm. (I) -(J), Quantitative readout of primary root meristem size (I), and mature cortical cell length (J). Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 20 plants. Different letters represent significant differences at P<0.05 according to Tukey's HSD test.

Figure

  Figure S2. Cytokinin signaling is not involved in ammonium-dependent inhibition of root growth. (A) Expression pattern of the cytokinin reporter TCS:GFP in primary root tips under different N supply. Images were taken 6 d after transfer. (B) Quantitative readout of TCS:GFP fluorescence intensity in primary roots under different N supply. Boxes show the first quartile, median and third quartile; the whiskers show minimum and maximum values, n = 10 plants. Asterisks denote significant differences between wild type and indicated lines at * P<0.05 by two-tailed Student's t-test. (C) and (G), Root phenotype of wild-type (Col-0), ahk3-3, arr1-3, arr12-1 mutants (C), and wild-type

Figure

  Figure S3. H2O2 aggravates root growth inhibition in the presence of ammonium. (A) Root phenotype of wild-type plants 6 d after transfer to different N supply, in the absence or presence of either 1 mM H2O2 or 20 µM SHAM. Horizontal marks along the root axis indicate daily positions of primary root tips. Root images were scanned 6 d after transfer. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) and under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control and indicated treatments at each time point as * P<0.05 ** P<0.01 *** P<0.001 according to Dunnett's multiple test. (D) Cell length and meristem size of primary roots. The length of mature cortical cells and the size of the root apical meristem are indicated by yellow arrowheads. DIC images of primary root tips were taken at 6 d after transfer. Scale bar = 100 µm. (E) -(F) Quantitative readout of cortical cell length (E), and meristem size (F). Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 16 plants. Different letters represent significant differences between samples according

Figure

  Figure S4. Superoxide is not causal for ammonium-dependent root growth inhibition. (A) Schematic diagram of ROS metabolism and inhibitor action. Sodium diethyldithiocarbamate (DDC) acts as an inhibitor of superoxide dismutase (SOD), potassium iodide (KI) acts as H2O2 scavenger, and salicylhydroxamic acid (SHAM) is a peroxidase inhibitor (Lee et al., 2013). (B) Root phenotype of wild-type plants 6 d after transfer to different N supply in the absence or presence of 100 µM DDC. Horizontal marks along the root axis indicate daily positions of primary root tips. Scale bar = 1 cm. (C) -(D) Relative primary root elongation rate under 1 mM ammonium supply (C) and under 10 mM ammonium supply (D), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control and DDC treatment at each time point according to two-tailed Student's t-test at *** P<0.001. (E) Cell length and meristem size of primary roots. Primary roots were stained by propidium iodide, and confocal images were taken 6 d after transfer. The length of mature cortical cells and the size of the root apical meristem are indicated by red arrowheads. Scale bar = 100 µm. (F) -(G) Quantitative readout of cortical cell length (F), and meristem size (G). Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 20 plants.

Figure S5 .

 S5 Figure S5. Screening of rboh mutants under different N supply. (A) Root phenotypes of wild-type plants and single or multiple rboh mutants in response to different N supply. After a pre-culture of 6 d, seedlings were transferred to medium containing either 1 mM nitrate or 1 mM ammonium as sole N source. Horizontal marks along the root axis indicate daily positions of primary root tips. Images were taken 6 d after treatment. Scale bar = 1 cm. (B) Primary root length after transfer. Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 18 plants. Different letters represent

Figure S6 .

 S6 Figure S6. Fe precipitation in the primary root under nitrate or ammonium supply.After a pre-culture of 6 d, wild-type plants were transferred to the medium supplied with 1 mM nitrate, 1 mM ammonium or 10 mM ammonium. Perls/DAB stained roots were embedded in Spurr resin. Root cross-sections were obtained from the differentiation zone of primary roots. Fe precipitation in primary roots is indicated by a reddish-brown colour. Scale bars: 50 µm.

Figure S7 .

 S7 Figure S7. Dynamic changes of the Fe and ROS status in primary root tips under different N supply. (A) Perls/DAB staining of Fe as indicated by a reddish-brown color. After a preculture of 6 d, wild type (Col-0) plants were transferred to the treatment medium supplied with differing N forms. Histological staining was performed at the indicated days after transfer (DAT). Scale bars: 100 µm. (B) ROS status (oxidant levels) in primary root tips as monitored by H2DCFDA staining. DCF fluorescence was detected by confocal microscopy at the indicated time points. Rainbow color code (black to white) indicates DCF fluorescence intensity (low to high). Scale bars: 100 µm. (C) Quantitative readout of the fluorescence intensity of H2DCFDA staining. The boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 12 plants. Different letters represent significant differences between samples at P<0.05 according to Tukey's HSD test. Orange arrowheads in (A), or white arrowheads in (B), indicate the boundaries of the meristematic zone, elongation zone and differentiation zone along the primary root.

Figure S8 .Figure

 S8 Figure S8. Fe aggravates ammonium-repressed cell division in roots. (A) GUS activity in primary root tips of a CycB1;1::GUS reporter line. After a preculture of 6 d, CycB1;1::GUS reporter plants were transferred to treatment medium supplied with different N forms. The treatment medium contained 100 µM Fe(III)-EDTA (control) or 10 µM Fe(III)-EDTA (low Fe). Histological staining was performed 6 d after transfer. Scale bar = 100 µm. (B) Quantitative readout of the dividing cell number in primary root tips of CycB1;1::GUS reporter line. Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 12 plants. Different letters represent significant differences between samples according to two-way ANOVA followed by Tukey's HSD test at P<0.05.

Figure S10 .

 S10 Figure S10. Changes in rhizosphere pH after supply of different N forms.After a pre-culture of 6 d, wild-type (Col-0) seedlings were transferred to the treatment medium supplied with different N forms. Medium pH of 5.0, 5.7 and 6.5 was buffered by 2.5 mM MES, while pH 7.2 was buffered by 2.5 mM MOPS.Changes of medium pH through plants are monitored by the pH indicator

Figure S11 .

 S11 Figure S11. Influence of buffer strength on root growth and root Fe status. (A) Root phenotype of wild-type (Col-0) plants 6 d after transfer to treatment medium containing MES buffer at different strength. Horizontal marks along the root axis indicate daily positions of primary root tips. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under supply of 1 mM ammonium (B) or 10 mM ammonium (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between 2.5 mM MES and indicated treatments at each time point as * P<0.05 ** P<0.01 *** P<0.001 according to Dunnett's multiple test. (D) Perls/DAB staining of Fe in primary root tips as indicated by a reddish-brown color. Perls/DAB staining was conducted at 6 d after transfer. Representative images from 10 plants per treatment are shown. Scale bars: 100 µm.

Figure

  Figure S12. Light is not essential for ammonium-dependent inhibition of primary root elongation. (A) Experimental setup of the root shading treatment. Petri dish-grown plants were exposed to white light in the control treatment (light), while the whole root zone was covered by aluminium foil in the shading treatment (shading), as described in Zheng et al. (2019). (B) Appearance of plants grown on the indicated nitrogen supply. After a pre-culture of 6 d, wild-type (Col-0) plants were transferred to the treatment plates and either exposed to light or shaded. Images were taken 6 d after transfer. Scale bar = 1 cm. (C) Primary root length after transfer of wild-type plants to light or shading. Boxes show the first quartile, median and third quartile; the whiskers show minimum and maximum values; n = 20 plants. Different letters represent significant differences at P<0.05 according to Tukey's HSD test.

Figure

  Figure S13. The formation of hydroxyl radicals is not critical for ammoniuminhibited root elongation. (A) Root phenotype of wild-type (Col-0) plants 6 d after transfer to different N supplies in the absence (control) or presence of 0.5 mM or 1.0 mM thiourea. Horizontal marks along the root axis indicate daily positions of primary root tips. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under supply of 1 mM ammonium (B) or 10 mM ammonium (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control and thiourea

Figure S14 .

 S14 Figure S14. pdx1.1 mutants are hypersensitive to ammonium. (A) Phenotypic screening of T-DNA insertion lines of ammonium-responsive genes. Primary root length under ammonium supply was taken as a read-out for ammonium sensitivity. Candidate lines were pre-cultured on half-strength MS medium containing 1 mM nitrate for 6 d before being transferred to medium containing either 1 mM nitrate or 1 mM ammonium as sole N source. Horizontal marks along the root axis indicate daily positions of primary root tips. Images were taken 6 d after transfer. Scale bar = 1 cm. (B) Relative primary root length after transfer to 1 mM ammonium, normalized to the root length of plants treated with 1 mM nitrate. Asterisks denote significant differences between wild-type (Col-0) and indicated lines as *** P<0.001 according to by Dunnett's multiple test; ns = not significant. (C) Schematic diagram of the T-DNA insertions in the PDX1.1 gene. Positioning of the T-DNA insertion in pdx1.1-3 (SALK_024245) and the transposon insertion in pdx1.1-1 (SM_3_22664) (Titiz et al., 2006). (D) Root phenotype of wild-type (Col-0), pdx1.1-1 and pdx1.1-3 plants subjected to different N supplies. Horizontal marks along the root axis indicate daily positions of primary root tips. Images were taken 6 d after transfer. Scale bar = 1 cm. (E) Primary root length of wild-type (Col-0) and pdx1.1 mutant plants after transfer to different N forms. Boxes show the first quartile, median and third quartile; the whiskers show minimum and maximum values; n = 18 plants. Different letters represent significant differences between samples according to two-way ANOVA followed by Tukey's HSD test, P<0.05.

Figure S15 .

 S15 Figure S15. Vitamin B6 metabolism in Arabidopsis.Vitamin B6 comprises six different vitamers. PLP, as a phosphorylated B6 vitamer is a bioactive form participating as coenzyme in numerous enzymatic reactions. Nonphosphorylated forms of vitamin B6 are PN, PL, and PM. In plants, PLP is produced by biosynthesis de novo (blue) or by the salvage pathway (brown) via the conversion among different B6 vitamers. This schematic is modified from[START_REF] Colinas | Balancing of B-6 vitamers is essential for plant development and metabolism in Arabidopsis[END_REF].

Figure S16 .

 S16 Figure S16. Exogenous application of vitamin B6 rescues cell length and meristem size under ammonium supply. (A) -(B) Cell length and meristem size of primary roots of wild-type (Col-0) (A) or pdx1.1-3 mutant (B). Plants grown under different N supply and in the absence (-VB6) or presence (+ VB6) of 5 µM pyridoxine. DIC images of primary roots were taken 6 d after transfer. The length of mature cortical cells and the size of the root apical meristem are indicated by yellow arrowheads. Scale bar = 100 µm. (C) -(D) Quantitative readout of cortical cell length (C), and meristem size (D). Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 16 plants. Different letters represent significant differences between samples according to three-way ANOVA followed by Tukey's HSD test, P<0.05.

Figure S17 .

 S17 Figure S17. Exogenous application of vitamin B6 restores cell division under ammonium supply. (A) GUS activity of the CycB1;1::GUS reporter in primary roots of wild-type (Col-0) and pdx1.1-3 mutant plants. After a pre-culture of 6 d, CycB1;1::GUS reporter lines were transferred to different N forms in the absence (-VB6) or presence (+ VB6) of 5 µM pyridoxine. Histological staining were performed 6 d after treatment. Scale bar = 100 µm. (B) Quantitative readout of the number of dividing cells. Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 12 plants. Different letters represent significant differences among treatments according to three-way ANOVA followed by Tukey's HSD test at P<0.05.

Figure

  Figure S18. Non-phosphorylated forms of vitamin B6 are critical for ammonium detoxification. (A) Root phenotype of wild-type (Col-0) plants 6 d after transfer to different N supply in the absence or presence of 5 µM vitamin B6 provided either as pyridoxal 5'phosphate (PLP), pyridoxal (PL), or pyridoxine (PN). Horizontal marks along the root axis indicate daily positions of primary root tips. Seedlings subjected to N supplies with exogenous supplementation of different B6 vitamers. Scale bar = 1 cm. (B) -(C) Relative primary root elongation rate under 1 mM ammonium supply (B) or under 10 mM ammonium supply (C), normalized to the growth rate of plants treated with 1 mM nitrate. Symbols represent means ± SE, n = 20 plants per treatment. Asterisks denote significant differences between control and indicated treatments at each time point as * P<0.05 ** P<0.01 *** P<0.001 according to Dunnett's multiple test. (D) DAB staining of H2O2 in primary root tips 6 d after treatment. The reddish-brown coloration

Figure

  Figure S19. Over-expression of PDX1.1 rescues cell division under ammonium supply. (A) GUS activity of the CycB1;1::GUS reporter in primary root tips of wild-type (Col-0), pdx1.1-3 and PDX1.1OE-L5 plants. After a pre-culture of 6 d, CycB1;1::GUS reporter lines were transferred to the treatment medium supplied with different N forms. Histological stainings were performed 6 d after transfer. Scale bar = 100 µm. (B) Quantitative readout of the number of dividing cells in primary root tips of CycB1;1::GUS reporter lines. Boxes show the first quartile, median and third quartile; the whiskers show the minimum and maximum values, n = 12 plants. Different letters represent significant differences between treatments and lines according to two-way ANOVA followed by Tukey's HSD test at P<0.05.

Figure S20 .

 S20 Figure S20. Influence of medium pH on the expression of PDX1.1 upon ammonium supply. Promoter activities of the pPDX1.1:GUS reporter in primary roots subjected to different N forms and buffered at different pH were monitored by GUS staining 6 d after transfer. Medium pH of 5.0, 5.7 and 6.5 was buffered by 2.5 mM MES, while pH 7.2 was buffered by 2.5 mM MOPS. Representative images from 10 plants per treatment are shown. Scale bar = 100 µm.

Figure

  Figure S21. Over-expression of PDX1.1 improves tolerance to P deficiency and Ni toxicity. (A) -(B) Heat map displaying the relative expression level of vitamin B6-related genes (highlighted in boxes) in response to phosphorus deficiency (A) or nickel toxicity (B). Data were retrieved from published microarray experiments (Bhosale et al., 2018; Lesková et al., 2020). Selected genes previously shown to be up-or downregulated by phosphorus deficiency or nickel toxicity were included as controls. -P, 5 µM phosphate; +P, 625 µM phosphate; ++Ni, 100 µM nickel; control, no nickel added to growth medium. (C) Appearance of plants grown on different phosphate (P) supplies. After a pre-culture of 6 d, the seedlings of Col-0, pdx1.1-3, PDX1.1OE-L5, PDX1.1OE-L15 and PDX1.1OE-L8 were transferred to treatment medium containing 625 µM P (+P) or 5 µM P (-P). Images were taken 6 d after transfer. Scale bar = 1 cm. (D) Primary root length after transfer. Root phenotyping of indicated lines was conducted 6 d after transfer. Boxes show the first quartile, median and third quartile;

ACKNOWLEDGMENTS

We thank Twan Rutten (IPK Gatersleben) for microscopy advice. We thank Markus Meier, Cevza Esin Tunc, and Mohammad-Reza Hajirezaei (IPK Gatersleben) for scientific advice and valuable discussions. We further thank Jacqueline Fuge, Annett J o u r n a l P r e -p r o o f Bieber, Elis Fraust, Barbara Kettig, Yudelsy Antonia Tandron Moya, Dagmar Böhmert and Christine Bethmann (IPK Gatersleben) for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Germany) with a grant (WI1728/13-2) to N.v.W. The Swiss National Science Foundation is also acknowledged for funding to T.B.F. (grants 31003A_14117 and IZLIZ3_183193).