Modulation of ethylene biosynthesis by ACC and AIB reveals a structural and functional relationship between the K 15 NO 3 uptake rate and root absorbing surfaces

The modification of root traits in relation to nitrate uptake represents a source for improvement of nitrogen uptake efficiency. Because ethylene signalling modulates growth of exploratory and root hair systems more rapidly (minutes to hours) than nitrate signalling (days to weeks), a pharmacological approach was used to decipher the relationships between root elongation and N uptake. Rape seedlings were grown on agar plates supplied with 1 mM KNO3 and treated with different concentrations of either the ethylene precursor, ACC (0.1, 1, and 10 μM) or an inhibitor of ethylene biosynthesis, AIB (0.5 and 1 μM). The results showed that rapid modulation of root elongation (up to 8-fold) is more dependent on the ethylene than the nitrate signal. Indeed, ACC treatment induced a partial compensatory increase in N uptake associated with overexpression of the BnNRT2.1 and BnNRT1.1 genes. Likewise, daily root elongation between treatments was not associated with daily nitrate uptake but was correlated with N status. This suggested that a part of the daily root response was modulated by cross talks between ethylene signalling and N and C metabolisms. This was confirmed by the reduction in C allocation to the roots induced by ACC treatment and the correlations of changes in the root length and shoot surface area with the aspartate content. The observed effects of ethylene signalling in the root elongation and NRT gene expression are discussed in the context of the putative role of NRT2.1 and NRT1.1 transporters as nitrate sensors.


Introduction
The improvement of macronutrient acquisition efficiency (such as N, P, and K) in low-fertility soils constitutes one of the major goals of breeding programmes aimed at reducing fertilizer inputs and increasing nutrient use efficiency (Hirel et al., 2007;Lynch, 2007).As a consequence, in the next few years, traits responsible for root architecture changes will be one of the most important targets for plant breeders to improve soil exploration and nutrient acquisition.
Despite a clearly established role for ethylene in auxin synthesis, transport, and partitioning along the root (Stepanova et al., 2005;Ruzicka et al., 2007;Swarup et al., 2007), some studies have been carried out to measure the impact of pharmacological treatments or genetic modifications on ethylene synthesis and signalling during the acquisition of macronutrients such as nitrate (Leblanc et al., 2008;Beauclair et al., 2009;Tian et al., 2009).These genetic and pharmacological studies highlight the regulatory role of ethylene in nitratedependent root development by modulating the expression of the NRT1.1 and NRT2.1 nitrate transporters.Thus, etr1-3 and ein2-1 mutants of ethylene signalling show insensitivity to high nitrate concentrations (Tian et al., 2009).These results were confirmed by pharmacological treatment with the ethylene precursor, 1-aminocyclopropane-1 carboxylic acid (ACC), and with an inhibitor of ACC synthase (ACS), aminoethoxyvinylglycine (AVG), which are used to modulate ethylene biosynthesis and signalling.However, compared with a mutant approach, results obtained by a pharmacological approach have to be considered with caution because secondary targets or products of the inhibitors and activators are also biologically relevant.
Thus, several authors have demonstrated that inhibitors of ACS such as AVG impair not only ethylene synthesis but also auxin synthesis and amino acid metabolism (Leblanc et al., 2008;Beauclair et al., 2009;Soeno et al., 2010).Indeed, AVG is a suicide inhibitor of many pyridoxal phosphate-dependent enzymes that belongs to subgroup I of aminotransferases, which includes ACS and Trp aminotransferase, the first enzyme of ethylene biosynthesis and the main enzyme in acetic indole β acid (AIA) biosynthesis from l-tryptophan, respectively (Werck-Reichhart et al., 1988;Soeno et al., 2010).The non-specific inhibition of ACS by AVG treatment has been demonstrated through restoration of on root elongation after adding 1 mM potassium glutamate to roots treated with AVG (Leblanc et al., 2008).Similar inhibition and restoration effects in root growth was also obtained by using methionine sulphoximine (MSX), another PLP-enzyme inhibitor, in cotreatment with 5 mM glutamine (Gifford et al., 2008).
Likewise, recent studies have shown that morphological modifications induced by treatment with ACC result from cyanide (HCN) production and its metabolic effects.Indeed, HCN is produced stoichiometrically during oxidation of ACC by ACC oxidase to form ethylene (Peiser et al., 1984;Wang et al., 2002;Piotrowski, 2008).Recent studies with the cys-c1 mutant of β-cyanoalanine synthase, involved in HCN detoxification, have demonstrated that HCN accumulation acts as a repressive signal for several genes that encode enzymes involved in cell-wall rebuilding during root elongation of root and root hair tips (Garcia et al., 2010).Thus, the possibility that changes induced by ACC treatment in the BnNrt2.1 and BnNRT1.1 transcription levels are in fact, side effects of HCN metabolism cannot be precluded (Leblanc et al., 2008;Tian et al., 2009).
Accordingly, simultaneous inhibition of both ethylene and nitrogen metabolism by AVG treatment does not provide a clear conclusion about changes to nitrate uptake and the expression of nitrate transporters following ethylene stimulation of root elongation.In order to avoid inhibition of nitrogen metabolism during AVG modulation of ethylene biosynthesis, the present study used another ethylene biosynthesis inhibitor: α-aminoisobutyric acid (AIB).This structural analogue of ACC inhibits specifically the activity of ACC oxidase (Satoh and Esashi, 1982;Liu et al., 1984;Xu et al., 2008;Tsang et al., 2010) and avoids the production of ethylene and HCN from ACC (Peiser et al., 1984;Piotrowski, 2008;Garcia et al., 2010).
This study investigated to what extent root architecture modification (structure) correlates to nitrate uptake (function).It examined whether the elongation changes of exploratory and root hair systems induced by ethylene affects nitrate uptake and the expression of BnNRT nitrate transporter genes.The results clearly demonstrate that ethylene signalling is involved in nitrate uptake regulation in relation to root elongation changes.They also reveal a specific relationship between changes in aspartic acid concentrations induced by ACC in the roots and shoots and variations in the root length and N status.

Plant material and growth conditions
Rape (Brassica napus L. cv.Capitol) seeds were treated with 70% alcohol for 3 min, followed by four rinses in sterile water and were placed in Petri dishes (12 × 12 cm) containing filter paper (Whatman 3M) saturated with sterile water and incubated for 48 h in the dark at room temperature (22 °C).The germinated seeds were then sorted according to radicle length (5-6 mm) and four seedlings were selected and transferred onto new Petri dishes containing 50 ml solidified agar culture medium with different chemical treatments.The basic medium (pH 6.75) contained 0.4 mM KH 2 PO 4 , 0.15 mM K 2 HPO 4 , 1 mM K 2 SO 4 , 0.5 mM MgSO 4 , 3 mM CaCl 2 , 0.2 mM Fe-Na EDTA, 14 μM H 3 BO 3 , 5 μM MnSO 4 , 3μM ZnSO 4 , 0.7 μM CuSO 4 , 0.7μM (NH 4 ) 6 Mo 7 O 24 , and 0.1 μM CoCl 2 and was solidified with 0.8% (w/v) agar (A-7002, Sigma-Aldrich).This medium was supplemented with 1 mM KNO 3 as a sole nitrogen source for all treatments.ACC (A 3903, Sigma-Aldrich) and AIB (850993, Sigma-Aldrich) were dissolved in sterile water to a final stock concentration of 10 mM.Adequate volumes of chemicals were added to 50 ml autoclaved cultured medium with 1 mM KNO 3 in a falcon tube, mixed and then poured into Petri dishes under laminar sterile airflow.After transfer of 2-day-old germinated seedlings, Petri dishes were half sealed with adhesive tape.The dishes were placed vertically in a growth chamber at 22 °C under a 16/8 light/dark regime with a light intensity of 200 μmol m -2 s -1 .

Root system length and cotyledonary surface area morphometric analyses
The daily elongation of the primary root apex was measured on treated seedlings with different concentrations of AIB (0.5 and 1 μM) and ACC (0.1, 1, and 10 μM) in the presence of 1 mM KNO 3 Downloaded from https://academic.oup.com/jxb/article-abstract/64/10/2725/541440/Modulation-of-ethylene-biosynthesis-by-ACC-and-AIB by guest on 15 September 2017 between 48 and 72 h of treatment as described by Leblanc et al. (2008).Transparent slides were placed onto the back of the Petri dishes to record the daily elongation of primary roots by marking the slide with a pencil.Then, for each treatment, the daily elongation of the primary root length was measured from the slides with a WinRHIZO scan system (Regent Instruments, Quebec, Canada).
The effects of treatment on the exploratory root system (primary and lateral roots) and shoot area were measured every day during a kinetic study spanning 5 days.For each pharmacological treatment, four repeats corresponding to four different agar plates composed of four seedlings were harvested.The root and shoot parts of the seedlings were excised.Seedling roots were washed in a 1 mM CaSO 4 solution for 1 min at room temperature before being placed in demineralized water and analysed with the WinRHIZO scan system.The shoot parts were laid on a transparent slide and photocopied (Kyocera Mita copier, KM-2030) to further quantify the leaf area with the WinRHIZO scan system.After these treatments, extra water was removed from the roots and shoots and they were dried on tissue paper and placed into 2-ml Eppendorf tubes.After weighing to obtain the fresh weights, roots and shoots were dried in an oven for 72 h at 60 °C then weighed to get their dry weight.Before isotope analyses, the shoots and roots were separately ground for 2 min to a fine powder with a 4-mm inox ball in an oscillating grinder at 30 s -1 frequency (Retsch mixer mill, MM301, Haan, Germany).

Net K 15 NO 3 -uptake and isotope analysis
Net uptake of NO 3 -was obtained by 15 N labelling.For each point of the kinetic study (24, 48, 72, 96, and 120 h), the medium was supplemented with K 15 NO 3 (atom % 15 N: 1%).The total 15 N amount was determined for roots and shoots.The 15 N analyses were performed using an analyser (EA 3000, Eurovector, Milan, Italy) coupled with an isotopic mass spectrometer (IsoPrime X, GV Instruments, Manchester, UK).

RNA isolation and quantitative RT-PCR analyses
For gene expression analysis, total RNA was extracted after 5 days of treatment from four seedlings each (200-400 mg of root fresh matter) sampled from three-five sets of experiments with the different ACC and AIB treatments.Fresh root samples frozen in 2-ml Eppendorf tubes in liquid nitrogen were ground for 1 min 30 s with a 4 mm steel ball in an oscillating grinder at 30 s -1 (Retsch mixer mill MM301) before the extraction of total RNA with an RNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions.Then, total RNA was treated with DNase I (Qiagen) before quantification.Quantification was performed with a spectrophometer at 260 nm (BioPhotometer, Eppendorf, Le Pecq, France) before RT-PCR analyses.For RT, 1 μg total RNA was converted to cDNA with an iScript cDNA synthesis kit (Bio-Rad, Marne-la-Coquette, France) using the manufacturer's protocol.Gene expression levels were normalized to the expression level of the 18S gene (house-keeping gene) (forward (F), 5'-CGGATAACCGTAGTAATTCTAG-3' and reverse (R), 5'-GTACTCATTCCAATTACCAGAC-3').The primers used to amplify the specific gene sequences were for BnNrt2.1 F 5'-TGGTGGAATAGGCGGCTCGAGTTG-3' and R 5'-GTATACGTTTTGGGTCATTGCCAT-3' (AJ293028) and for BnNrt1.1 F 5'-ATGGTAACCGAAGTGCCTTG-3' and R 5'-TGATTCCAGCTGTTGAAGC-3' (AJ278966).The subsequent PCR reactions were performed with 4 μl of 200× diluted cDNA, 500 nM of the relevant primer, and 1× SYBR Green PCR Master Mix (Bio-Rad) in a final volume of 15 μl.The specificity of PCR amplification was examined by monitoring the melting curves after quantitative PCR reactions using a Chromo4 System (Bio-Rad) and by sequencing the amplification products to confirm that the correct amplicons were produced from each pair of primers (Biofidal, Vaulx en Velin, France).The amplification efficiency for each amplicon (18S, 103%; NRT2.1, 93%; and NRT1.1, 96%) was obtained according to the CFX Manager Software (Biorad) based on the Cq method of Rasmussen (2001).Comparative relative expression of the various genes was determined using the delta-delta method employing the formula relative expression = 2 -[ΔCt sample-ΔCt control]  where Ct refers to the threshold cycle, sample indicates the gene of interest, and control indicates the endogenous housekeeping gene (Livak and Schmittgen, 2001).

Amino acid profiling
Amino acid profiling was performed on shoot and root material using the ACQUITY UltraPerformance LC (UPLC) separation system (Waters, Milford, USA) according to Renault et al. (2010).Seedling tissues sampled from two different experiments with AIB and AVG were collected, freeze-dried and homogenized with a 4-mm inox ball for 1 min at 30 s -1 frequency.Methanol/chloroform/water-based extractions were made from 10 mg of the resulting dry powder.The powder was suspended in 400 μl methanol containing 200 μM dl-3-aminobutyric acid (BABA) as an internal standard and agitated at 1500 rpm for 15 min at room temperature.Chloroform (200 μl) was then added and samples were agitated at 1500 rpm for 5 min at room temperature.Ultra-pure water (400 μl) was added and samples were vigorously mixed and then centrifuged at 13,000 g for 5 min at 4 °C.The upper phase containing amino acids was transferred to a clean microtube, dried under vacuum, and the dry residue was resuspended in 600 μl ultra-pure water.A 5 μl aliquot of the resulting extract was used for derivatization according to the AccQ.Tag Ultra Derivatization Kit protocol (Waters) and then derivatized amino acids were analysed using an ACQUITY UPLC system.An aliquot (1 μl) of the reaction mix was injected onto an ACQUITY UPLC BEH C18 1.7 μm 2.1 × 100 mm column heated at 55 °C.Elution of amino acids was performed with a mix of 10-fold diluted AccQ.Tag Ultra Eluent (A) and acetonitrile (B) at 0.7 ml min -1 flow according to the following gradient: initial, 99.9 % A; 0.54 min, 99.9 % A; 6.50 min, 90.9 % A, curve 7; 8.50 min, 78.8 % A, curve 6; 8.90 min, 40.4 % A, curve 6; 9.50 min, 40.4 % A, curve 6; 9.60 min, 99.9 % A, curve 6; 10.10 min, 99.9 % A. Amino acids were detected at 260 nm using a photo diode array detector and were subsequently identified and quantified with the individual external standard calibration curves.

Statistical analyses
For each tested treatment, values are the mean ± SE of four replicates corresponding to four agar plates of four seedlings each.Statistical analyses were performed on Minitab statistical software version 13.2.The data were analysed by the nonparametric test of Kruskall-Wallis.Then, the Mood median tests were used to compare means or medians; bars sharing different letters are significantly different at P = 0.05 (Sokal and Rohlf, 2003).The regression coefficients were given by Excel 2008 version 12.0.The significance for each regression coefficient was obtained for DF = N-2 in Table A11 of Snedecor and Cochran (1957).

ACC and AIB applications modify elongation of the primary root and the exploratory root systems
Changes in the primary and exploratory root systems were examined on 2-day-old seedlings growing on agar medium containing 1 mM K 15 NO 3 after short-(24 h) or long-term treatment (120 h) with different concentrations of the ethylene precursor, ACC, or an inhibitor of ethylene biosynthesis, AIB (Fig. 1).Because ACC and AIB are supposed to induced opposite effects on elongation of the exploratory and root hair systems by modulating the ethylene endogenous content found in control conditions (1 mM KNO 3 treatment), KNO 3 treatment (used as control) was systematically placed between the AIB and ACC treatments in all the graphs.After 24 h of ACC treatment, elongation of primary root length was reduced in a dose-dependent way, whereas treatment with AIB did not significantly change primary root length compared to the control (Fig. 1A, B).
However, after 120 h of application, the effects of both treatments were more pronounced on root elongation and shoot expansion (Fig. 1C).Compared to control, the length of the exploratory root system (primary and lateral roots) was slightly increased in AIB-treated seedlings, whereas it was significantly reduced in ACC-treated seedlings (Fig. 1C).Because of the one order of magnitude difference in root elongation between AIB and ACC treatments, ethylene modulation by ACC and AIB allows study of the impact of root architecture modification (structure) on nitrate uptake (function).

AIB treatment has no effect on N metabolism compared to AVG
In order to check if long-term (120 h) AIB treatment had no effect on amino acid (AA) metabolism, AA profiles were compared between control and treatments.The comparison also included AA profiles of AVG-treated seedlings because it was previously reported that AVG inhibited pyridoxal enzymes and/or aminotransferases and significantly modified the AA profile (Leblanc et al., 2008;Beauclair et al., 2009).The comparison of root and shoot AA profiles between KNO 3 (control) and AIB (1 μM) or AVG (10 μM) treatments from two independent experiments demonstrated that AIB had no effect on root and shoot AA metabolism, unlike AVG (Table 1 and Supplementary Table S1, available at JXB online).Moreover, as the effects of AVG on AA profiles were more pronounced in the root than in the shoot, this suggests that AVG cannot reach the shoot (Table 1 and Supplementary Table S1).
Accordingly, the use of AIB allowed a more accurate study of the impact of ethylene metabolism modulation on changes to N metabolism because it had no direct side effects on N metabolism such as the endogenous AA content of the shoots and roots.Likewise, AIB avoids the strong anti-auxin activity induced by AVG treatment (Soeno et al., 2010) and the inhibitory effect of AVG on primary and lateral root elongation (Leblanc et al., 2008).

Ethylene biosynthesis modulation by ACC and AIB affects C partitioning between roots and shoots
To further investigate how the modulation of ethylene biosynthesis by ACC and AIB might affect the allocation of carbon in relation to morphological changes in the shoots and roots, patterns of C accumulation in the roots and shoots were compared to the organ dry weight between treatments (Fig. 2).The results showed that the stimulation of ethylene biosynthesis by ACC treatment caused a greater reduction in the pattern of C and dry weight allocation to the root (Fig. 2B) than to the shoot (Fig. 2A).Accordingly, high and significant correlation was found between C accumulation and root length (Supplementary Fig. S1).
As external nitrate concentration in the agar medium was similar for all ACC and AIB treatments (1 mM KNO 3 ), it can be reasonably assumed that changes to the root architecture induced by ethylene biosynthesis modulation were mainly associated with the impairment of C translocation from the shoots to roots.In contrast, dry weight and C accumulation Fig. 1.Effects of ACC and AIB on primary root and exploratory root system elongation after 24 h and 120 h of treatment in Brassica napus plantlets grown on agar plates under homogeneous feeding of KNO 3 (1 mM).(A) Dose-response curves of ACC or AIB treatments on daily elongation of the primary roots between 48 h and 72 h treatment.(B) Effects of AIB and ACC treatments on the correlation established between primary root length and daily elongation of the primary root.Each bar represents the mean length per treatment ± SE for n = 6-20 seedlings.(C) Impact of ACC and AIB treatments on the exploratory root system (primary and lateral roots) and shoot surface area after 120 h of treatment.Values are mean ± SE of four repeats of four seedlings each.
in the shoots were less affected by modulation of ethylene biosynthesis except under 1 and 10 μM ACC concentrations (Fig. 1C and Fig. 2A).This led to a significant decrease in the ratio of leaf-specific area (ratio of the leaf surface area per leaf dry weight, Supplementary Fig. S2).

N uptake
The impact of root elongation changes induced by the modulation of ethylene synthesis on N accumulation and allocation between the shoots and roots was obtained by using K 15 NO 3 labelling during the 5 days of treatment (Fig. 3).Logarithmic relationships were established every day for the different pharmacological treatments between the content of 15 N taken up in the seedling and the length of the exploratory root system during the time course of the experiment (Fig. 3A).The results revealed that after 5 days of treatment, AIB-treated seedlings accumulated more 15 N, whereas ACC-treated seedlings accumulated less 15 N than control (Fig. 3A).However, the relationship between 15 N uptake rate (expressed as μmol h -1 cm -1 root) and total root length during the time course of experiment revealed that the longest root lengths induced by AIB and KNO 3 had the lowest nitrate uptake rate whereas the lowest root lengths induced by ACC were partially overcompensated by the 15 N uptake rate (Fig 3B).In addition, although increasing ACC concentrations significantly reduced the 15 N content in the shoots and roots after 5 days of treatment (Supplementary Fig. S3), 15 N allocation between the shoots and roots remained unchanged up to the 0.1 μM ACC treatment (Fig. 3C).All these results demonstrated that modulation of ethylene metabolism exerted a direct or indirect control on nitrate uptake and accumulation in relation to shoot and root growth.

Reduction in root elongation by ACC treatment induces a compensatory increase in the 15 N uptake associated with overexpression of BnNRT genes
To allow a more complete characterization of the 15 N uptake rate in response to the root elongation changes that were induced by modulation of ethylene biosynthesis, the 15 N uptake rate per unit of root length was calculated after 5 days of pharmacological treatment.The expression of the BnNRT2.1 and BnNRT1.1 genes in roots was also compared after 5 days of treatment (Fig. 4).Despite an increase in total 15 N accumulation in AIB-treated plantlets, the 15 N uptake rate per unit root length was slightly enhanced compared to control (Fig. 4A).In contrast, in ACC-treated plantlets, the considerable reduction of the exploratory root elongation was partially compensated by a large increase in the 15 N uptake rate per unit of root length compared to control plantlets (Fig. 4A).Although this compensation effect was observed Table 1.Comparison of root amino acid contents in Brassica napus plantlets grown for 5 days on agar plates and treated with 1 mM KNO 3 (control) or 1 mM KNO 3 with 1 μM α-aminoisobutyric acid (AIB) or 10 μM aminoethoxyvinylglycine (AVG).AVG and ACC treatments correspond to two sets of experiments.Amino acids were extracted and analysed according to Le Ny et al. (2013) during the time course of the experiment (Supplementary Fig. S4), it was unable to restore growth and 15 N accumulation to control levels (Fig. 3A).In fact, this compensatory effect was explained by a significant increase in BnNRT2.1 and BnNRT1.1 transcript levels (Fig. 4B and Supplementary Fig. S5).Thus, in roots of plantlets grown with 0.1 mM ACC, BnNRT1.1 and BnNRT2.1 transcript levels were 2-and 4-fold higher compared to control plantlets.Although ACC treatment induced BnNRT1.1 and BnNRT2.1 transcript levels more than in control (Fig. 4B and Supplementary Fig. S5), the highest transcript levels obtained for 0.1 μM ACC were not related to the highest 15 N uptake rate (Fig. 4A).This suggested that a differential regulation of BnNRT1.1 and BnNRT2.1 transporters might exist between transcription and activity (Fig. 4A and B).However, this discrepancy was not explained by specific changes in amino acid contents in the roots and shoots after 120 h of ACC treatment.Indeed, the endogenous contents in Gln and Glu in the roots and shoots were unchanged during the decrease in BnNRT1.1 and BnNRT2.1 expression levels and the increase in the 15 N uptake rate (Supplementary Fig. S6).

Relationships between structural and functional adjustments induced by modulation of ethylene metabolism are governed by the plant N status
The large range of changes in elongation of the exploratory root system (8-fold) after 5 days of treatment constitutes a unique tool to dissect root structural and functional relationships.Thus, correlations between changes in the 15 N uptake rate and root elongation were sought systematically (Fig. 5).A significant exponential correlation was obtained between the daily elongation of the exploratory root system and the total 15 N uptake rate between treatments (Fig. 5A).This relationship was in line with the previous correlation found between the total 15 N uptake rate in the seedlings and the variation of total root length during the course of the experiment (Fig. 3B).This demonstrates that root structural and functional adjustments induced by ethylene metabolism modulation are smoothly coordinated and regulated through internal crosstalk.In order to investigate the effects of short-term  root elongation changes on the 15 N uptake rate, the daily root elongation and daily 15 N uptake rates during the course of the experiment were examined between treatments.No correlation was found between treatments (Fig. 5B).However, the daily increase in root elongation was significantly correlated with the 15 N content in seedlings during the previous days of treatment (Fig. 5C).For example, the daily root elongation between 24-48 h and 48-72 h was correlated with 15 N content during 0-24 h and 0-48 h, respectively.Accordingly, these results demonstrated that the potential for root elongation was associated with the plant N status, which was previously modulated by the effects of ethylene on plant growth and root absorption.

The ethylene-induced variations in root and shoot expansion correlate with aspartate concentration changes and N status
To investigate further the possible relationships between nitrogen and hormone metabolism in the responses of root length and shoot surface area to ethylene biosynthesis modulation, this study examined the changes in free amino acid contents in the roots and shoots.Aspartate (Asp) was the only one whose content in the shoots and roots showed significant decline in response to ethylene treatments (Fig. 6A).Hence, this decline was strongly correlated with the total root length and shoot expansion (Fig. 6B).Because daily root elongation was correlated with the 15 N accumulated in plantlets during the previous days of treatment (Fig. 5C), correlations between N status and the endogenous content of Asp were also sought.This revealed tight relationships between Asp content and N status in the shoots and roots suggesting that a metabolic link exists between ethylene and N metabolism (Fig. 6C and Supplementary Fig. S7).

Discussion
Nitrate signalling and its implication for modulation of the root system elongation have been the subject of considerable research effort over the last 40 years in order to decipher the nitrate signalling cascade responsible for root growth (Scheible et al., 1997;Stitt and Feil, 1999;Zhang et al., 1999).The responses of the root system to nitrate have been studied with different experimental devices enabling vertical or horizontal heterogeneous nitrate supply to the roots such as the technique of using segmented filter paper systems (Drew et al., 1973), split root systems (Drew, 1975;Lainé et al., 1995;Scheible et al., 1997;Gansel et al., 2001), and segmented agar plate systems (Zhang et al., 1999;Remans et al., 2006a).All studies using these techniques have allowed characterization of short-term compensation mechanisms (minutes to hours) acting at the nitrate transport level (function) or long-term compensation mechanisms (days to week) involved at the structural level (root architecture).The discrepancy between structural and functional responses in terms of duration (minutes versus weeks) led to the study of the impact of rapid change in root exploratory and root hair systems on the nitrate uptake and gene expression of the nitrate transporters NRT1.1 and NRT2.1.Indeed, the lag period (days to weeks) in the root architectural response to nitrate treatment is inconsistent with the effect of a ligand on a receptor activating a physiological or metabolic response in a few minutes or hours (Iten et al., 1999;Jones and Sussman, 2009).Fig. 5. Relationships between root elongation and 15 N uptake or accumulation in Brassica napus seedlings treated over 5 days on agar plates with varying external concentrations of ACC and AIB under homogeneous feeding of 1 mM K 15 NO 3 .(A) Correlations between daily root elongation and the 15 N uptake rate.(B) Relationship between daily root elongation and the daily 15 N uptake rate.(C) Correlation between daily root elongation and 15 N accumulation in the days before the daily root elongation.For example, the daily root elongations between 24-48 h and 48-72 h were correlated with 15 N accumulation during 0-24 h and 0-48 h, respectively.Values are mean ± SE of four repeats of four seedlings each.
Downloaded from https://academic.oup.com/jxb/article-abstract/64/10/2725/541440/Modulation-of-ethylene-biosynthesis-by-ACC-and-AIB by guest on 15 September 2017 Moreover, contrary to AIA and ethylene signalling mutants, no mutants of macronutrient transporters have demonstrated direct effects on short-term modification of the root architecture (Orsel et al., 2004;Remans et al., 2006a,b).Likewise, the short-term metabolic effect of nitrate (minutes to hours) obtained from transcriptomic studies failed to uncover the nature of one or more nitrate receptors or sensors (Wang et al., 2000;Wang et al., 2001;Scheible et al., 2004;Gutiérrez et al., 2007;Krouk et al., 2009;Nero et al., 2009;Vidal et al., 2010).Because ethylene signalling modulates growth of exploratory and root hair systems more rapidly (minutes to hours) than nitrate signalling (Le et al., 2001;Stepanova et al., 2005;Ruzicka et al., 2007;Swarup et al., 2007;Leblanc et al., 2008;Tian et al., 2009), a pharmacological approach to modulate ethylene biosynthesis was used to decipher the relationships between root elongation and N uptake and metabolism.To summarize, the study question here was not whether ethylene is involved in the nitrate-dependent root development but rather, does the modulation of ethylene signalling impair nitrate uptake and N metabolism?
In fact, this pharmacological study provided several lines of evidence confirming that ethylene is not only involved in the short-term control of exploratory and root hair system elongation in B. napus seedlings, but is also directly or indirectly implied in the control of nitrate uptake regulation and the modulation of C and N metabolism.

ACC-induced reduction in root length causes an upregulation in BnNrt2.1 and BnNrt1.1 expression
The first set of evidence was provided by the fact that compared to AIB and control treatments, ACC induced an upregulation in BnNrt2.1 and BnNrt1.1 expression.This increase in transcript levels fits well with the increasing nitrate uptake rate per unit root length in ACC-treated plantlets, even if this compensatory effect was unable to restore normal shoot and root growth.These results are also in agreement with the linear correlation previously found between changes in root length and BnNRT2.1 expression levels in response to 10 μM AVG supply or changes in nitrate availability (Leblanc et al., 2008;Le Ny et al., 2013).Thus, as previously proposed, BnNRT2.1 could be essential for nitrate uptake, and its expression level and activity might adapt to the elongation changes of the exploratory root system induced by environmental cues (Leblanc et al., 2008(Leblanc et al., , 2013;;Le Ny et al., 2013).The fact that NRT2.1 is specifically located in cortical and epidermal cell layers of the mature roots of Arabidopsis and rice is also in line with this assumption (Chopin et al., 2007;Girin et al., 2007;Orsel et al., 2007;Feng et al., 2011).Similarly, the decrease in BnNrt2.1 expression with the increase in ACC concentrations from 0.1 to 10 μM suggested that BnNrt2.1 expression level might adapt to changes in the absorbing surface of the whole mature root (exploratory and root hair systems) by a regulatory mechanism that remains to be deciphered (Leblanc et al., 2013).This does not preclude the possibility that this mechanism may act via the ethylenesignalling cascade on BnNrt2.1 and BnNrt1.1 transcription levels, as proposed by Tian et al. (2009).

Modulation of ethylene biosynthesis caused impairment in C and N metabolism and significant changes in root elongation and shoot expansion
The second set of evidence indicating that ethylene is involved in the control of root length via its interactions with C and N metabolism is provided by the linear correlation between the variations in root elongation induced alongside changes in the C accumulated in the roots (r 2 = 0.95, P < 0.001).This result confirms that root growth is strongly dependent on the supply of C (Aguirrezabal et al., 1994;Muller et al., 1998;Freixes et al., 2002) and shows that the reduction in the root growth induced by ethylene affects carbon allocation to the roots (Yazdanbakhsh et al., 2011).Although reduction in C allocation to the roots can be attributed to the effects of ethylene on reducing photosynthesis (Ahmed et al., 2006;Tholen et al., 2007), it could also result from indirect effects of ethylene on N metabolism (e.g.nitrate reductase activity).It is possible that during ACC treatment, HCN produced by oxidation of ACC by ACC oxidase (Peiser et al., 1984;Pirrung, 1985;Wang et al., 2002) accumulated and inhibited nitrate reductase activity, as has been recently reported (Garcia et al., 2010;Yu and Zhang, 2012).
The 15 N uptake rate is strictly adjusted alongside modulation of the exploratory root system by ACC and AIB treatments The third set of evidence indicating the involvement of ethylene in the control of root length is provided by the fact that at constant external nitrate concentration (1 mM), the daily primary root elongation mainly depends on ethylene signalling rather than the daily N uptake rate (Figs.1A and  5B).This indicates and confirms that ethylene signalling instead of nitrate signalling, plays a major role in shortterm elongation of the exploratory and root hair systems (Le et al., 2001;Stepanova et al., 2005;Ruzicka et al., 2007;Swarup et al., 2007;Leblanc et al., 2008;Tian et al., 2009).Moreover, the pharmacological treatments with ACC and AIB revealed that a strict adjustment exists from short-to long-term treatments between root elongation induced by ethylene modulation and the 15 N accumulation rate.These structural and functional adjustments were observed during a very large elongation change in the exploratory root system (8 fold).Hence, the results question the hypothesis proposed in the literature of a role for NRT2.1 and NRT1.1 nitrate transporters as transceptors that respond to nitrate during the root growth (Remans et al., 2006a,b;Walch-Liu and Forde, 2008).Indeed, this hypothesis comes from the possible double function of some proteins as transporters and sensors as observed in yeast (Smith et al., 2003;Walch-Liu and Forde, 2008).However, the studies with the nrt2.1 and nrt1.1 mutants have been less demonstrative because of the temporal gap between the perception of the nitrate signal and significant responses in root elongation of the nrt2.1 and nrt1.1 mutants (9 to 12 days after nitrate treatment, Remans et al., 2006a,b;Walch-Liu and Forde, 2008).In contrast, the present results highlight the importance of metabolic cross talk between hormone and N metabolism, as revealed by recent transcriptomic and pharmacological studies (Leblanc et al., 2008(Leblanc et al., , 2013;;Nero et al., 2009;Tian et al., 2009;Vidal et al., 2010).
Furthermore, the results lead to the conclusion that even under a homogeneous nitrate supply, large root proliferation does not confer a large ecophysiological advantage to plant growth.This conclusion is in agreement with recent evidence that in long-term experiments with nitrate-and phosphaterich patches (Linkohr et al., 2002;Kembel and Cahill, 2005;Jansen et al., 2006;de Kroon et al., 2009) and in long-term field experiments between N fertilized and unfertilized plants (Petersen, 1995;Gabrielle et al., 1998a,b) N treatments have little influence on the final root length and growth.
How the modulation of ethylene signalling impairs nitrate uptake and N metabolism?
The fourth set of evidence indicating that ethylene is involved in the control of N metabolism regulation is more indirect.Indeed, daily root elongation is also correlated to N status, which is calculated as total N accumulated during the previous days, suggesting that a part of the daily root response induced by ethylene resulted from the long-term effect of the cross talk between ethylene with N uptake and metabolism.In this way, the linear correlations found between Asp contents in the shoots and roots and total root length and N status is very intriguing.The results establish for the first time that a metabolic relationship exists between changes in N metabolism and variations in the root elongation.Thus, Asp could be considered as a marker of root elongation changes during ethylene biosynthesis modulation.The occurrence of a correlation between the root length and some individual AA does not imply causality.Because of many connections between metabolism and growth may be mediated by signalling pathways that impinge on developmental processes, it is difficult to draw inferences about changes in AA levels.However, Asp is known to plays a central role C/N balance (Coruzzi, 2003), auxin catabolism (Ludwig-Müller, 2009) and mutants defective in cytosolic aspartate aminotransferase (AAT) showed similar reductions in root growth (Miesak and Coruzzi, 2002;Leasure et al., 2011).Moreover, ACC synthase presents an active catalytic site very similar to AAT that shares the same function as in the AAT counterpart (Huang et al., 1991;Capatini et al., 1999).Hence, Asp represents a very serious target in the interaction between N and ethylene metabolism.
A key priority for future research will be to investigate the direct or indirect influence of ACC on nitrate uptake regulation in relation to changes in root absorbing surfaces (exploratory and root hair systems).Clear challenges will be to elucidate the possible interactions between ethylene metabolism and signalling with N metabolism via AIA homeostasis, AAT inhibition, or HCN production.

Fig. 3 .
Fig.3.A) Relationship between cumulative 15 N uptake in seedlings and total root length.(B) Cumulative amount of 15 N in the roots and shoots after 120 h of treatment with ACC and AIB under homogeneous feeding of 1 mM K 15 NO 3 .(C) Percentage of 15 N translocated from roots to shoots between the treatments.Values are mean ± SE of four repeats of four seedlings each.

Fig. 4 .
Fig. 4. (A) Impact of ACC and AIB on the 15 NO 3-uptake rate per cm of root length after 120 h of treatment.(B) Relative evolution compared to control (seedlings treated with 1 mM K 15 NO 3 ) of total root length, 15 N accumulation, and BnNrt2.1 and BnNrt1.1 transcript levels.Relative evolution = (influx of ACC-or AIB-treated seedlings -influx of control (KNO 3 ) seedlings) / influx of control (KNO 3 ) seedlings.

Fig. 6 .
Fig. 6. (A) Changes in the Asp content induced by ACC and AIB treatments in the shoot and root tissues of Brassica napus seedlings treated over 5 days on agar plates under homogeneous feeding of 1 mM K 15 NO 3 .(B) Relationships between the exploratory root length and shoot surface area with the Asp content in the roots and shoots.(C) Relationship between 15 N accumulation in the roots and shoots and Asp content in the roots.Values are mean ± SE of three or four repeats of four seedlings each.
. Values are mean ± SE μmol (g root dry weight) -1 of three repetitions of a set of four individual seedlings.Values sharing different letters are significantly different at P <0.05 (t-test) Fig. 2. Changes in dry weight (DW) and C accumulation in the shoot (A) and root (B) induced in Brassica napus plantlets co-treated over 120 h with ACC and AIB under homogeneous feeding of nitrate (1 mM).Values are mean ± SE of four repeats of four seedlings each.