Maize growth conditions
Maize seeds (Zea mays L., cv. PR33T56, Pioneer Hi-bred Italia S.p.A., Parma, Italy) were germinated on a plastic net placed at the surface of an aerated 0.5 mM CaSO4 solution in a growth chamber at 25°C in the dark. After 3 days, the seedlings were transferred into an aerated hydroponic system containing 0.5 mM CaSO4 under controlled climatic conditions: day/night photoperiod, 16/8 h; light intensity, 220μMol m-2-s-1; temperature (day/night) 25/20°C; relative humidity 70 to 80%. After 2 days (5-days-old) plants were transferred for a maximum of 24 h in a N-free nutrient solution containing (μM): KCl 5; CaSO4 500; MgSO4 100; KH2PO4 175; NaFe-EDTA 20; H3BO3 2.5; MnSO4 0.2; ZnSO4 0.2; CuSO4 0.05; Na2MoO4 0.05. N was supplied in the form of 1 mM CO (NH2)2 (urea-treated plants); or as control, plants were exposed to a N-free nutrient solution (control-plants). The pH of solution was adjusted to pH 6.0 with potassium hydroxide (KOH).
For the experiments of 15[N]-urea acquisition, urea-treated plants were exposed to nutrient solution containing 1 mM 15[N]-urea (98 atom% 15[N]; ISOTEC® Stable Isotopes, Sigma Aldrich, Milano, Italy).
Measurement of net high-affinity urea uptake in maize plants
After 4 hours from the beginning of the N-treatment, roots of intact seedlings were immersed for 10 min, a time span during which uptake remained linear, in 40 ml of a constantly stirred and aerated solution containing 500μM CaSO4 and up to 300μM urea (2.5, 5, 10, 25, 50, 100, 200 or 300μM urea). For each urea concentration, the uptake rates were determined using six urea-treated and six control-plants. Net uptake rate was measured as urea depletion from the solution per unit of time. Thus, samples of the solution (60μl) were taken every 2 min and the urea content was determined by diacetylmonoxime and thiosemicarbazide colorimetric assay (modified from Killingsbaeck [28]). Therefore a 60μl aliquot was mixed thoroughly with 120μl of colour development reagent, which consisted of 1:1 mixed colour reagent [7% (v/v) 0.2 m diacetylmonoxime; 7% (v/v) 0.05 m thiosemicarbazide]: mixed acid reagent [20% (v/v) sulphuric acid (H2SO4); 0.06% (v/v) 74 mM ferric chloride hexahydrate in 9% (v/v) ortho-phosphoric acid]. The samples were incubated for 15 min at 99°C (lid temperature: 105°C) in a thermocycler. The samples were cooled 5 min on ice and the urea concentration was determined spectrophotometrically by measuring the absorbance at 540 nm using a microtiter plate reader. The uptake rates were expressed as μMol urea g-1 root FW h-1.
Kinetic parameters of the high-affinity urea uptake system (Vmax and Km) were calculated in the 2.5-300μM concentration range by NonLinear Regression-Global Curve Fitting and the statistical analysis was performed by Normality Test (Shapiro-Wilk) using SigmaPlot 12.0 (Systat software, Point Richmond, USA).
Determination of urea concentration
Root and leaf urea concentrations were measured in time-course (up to 24 hours of treatment) by colorimetric assay as described above (modified from Killingsbaeck [28]). Approximately 100 mg (fresh weight) of freeze plant tissues were milled and suspended in 1 ml of water at 99°C for 3 min. After centrifugation at 15000 g for 2 min, 60μl of supernatant were incubated with 120μl of colour-development reagent as previously described. Kojima et al.[18] reported that ureides allantoin, ornithine, arginine and uric acid did not interfere with the urea determination by diacetylmonoxime and thiosemicarbazide.
N]-analysis
Approximately 1 mg of dried root and leaf tissues was transferred into a tin capsule for measurement of δ15N in one run. The analysis was carried out using a Delta V isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a Flash EA 1112 Elemental Analyser (Thermo Scientific, Bremen, Germany). The isotope ratios were expressed in δ ‰ versus air for δ15N according to the following formula: δ ‰ = [(Rsample-Rstandard)/Rstandard] ⋅ 1000 where Rsample is the isotope ratio measured for the sample and Rstandard is the isotope ratio of the international standard. R is the abundance ratio of the minor, heavier isotope of the element to the major, lighter isotope, as 15 N/14 N. The isotope values were calculated against international reference materials: L-glutamic acid USGS 41, ammonium sulphate IAEA-N-2 (IAEA-International Atomic Energy Agency, Vienna, Austria) and urea 33802174IVA (IVA Analysentechnik e.k.). The uncertainty of the nitrogen isotopic determination was ± 0.3‰.
Molecular work
RNA extraction
Total RNA was isolated from roots and leaves of maize plants. The RNA extractions were performed using the Invisorb Spin Plant RNA kit (Stratec Molecular, Berlin, Germany) as reported in the manufacturer’s instructions (http://www.invitek.de/). The integrity of RNA was qualitatively checked on a 1% agarose gel and quantified by spectrophotometer Nanodrop 2000 instrument (Thermo Scientific, Wilmington, USA).
Real-time RT-PCR experiments
One μg of total RNA was retrotranscribed in cDNA using Oligo-dT23 and the Superscript II Reverse Transcriptase (Gibco BRL, Basel, Switzerland), a RNase H derivative of moloney murine leukemia virus, according to the manufacturer’s protocol. After RNA digestion with 1 U RNase A (USB, Cleveland, USA) for 1 h at 37°C, gene expression analyses were performed by adding 0.16μl of the cDNA to the real-time RT-PCR complete mix, FluoCycle™ sybr green (20μl final volume; Euroclone, Pero, Italy), in a DNA Engine Opticon Real Time PCR Detection (Biorad, Hercules, USA).
Based on a ZmDUR3-EST sequence (BQ164112), specific primers (Tm = 58°C) were designed to generate 109 bp PCR product: CCTCAATCTGGTGGGTGTCT and ATTGGCCTTTCTCCACAGC (PCR efficiency 81%). Real-time RT-PCR analyses were performed in triplicates on three independent experiments. The analyses of real-time result were performed using Opticon Monitor 2 software (Biorad) and R (version 2.9.0; http://www.r-project.org/) with the qPCR package (version 1.1-8; [29]). Efficiencies of amplification were calculated following the authors’ indications [29]. Data were normalized with respect to the transcript level of the housekeeping gene (ZmRPS4, AF013487, GCAACGTTGTCATGGTGACT and CTCCACGTGAATGGTCTCAA, PCR efficiency 86%) using the 2-ΔΔCT method, where ΔΔCT = (CT,Target - CT,HK)Time x - (CT,Target - CT,HK)Time 0[30].
ZmDUR3-ORF cloning
In order to clone ZmDUR3-ORF, two reverse transcription reactions (RT-reaction) were performed, one reaction was transcribed using Oligo-dT23 while in the other reaction a specific primer for the ZmDUR3-ORF was used (2μM; reverse 5’-CAGGAATGAGGTGAAGAGCGCGAAGAAGGCGC-3’). For each reaction, 2 μg of total RNA were reverse transcribed.
Since the first 200 bp of the predicted ORF sequence were high in GC%, the ZmDUR3-ORF was amplified in two separate PCR-reactions; i.e. generating two fragments with an overlap of 20 bp, which were subsequently assembled using Assembly-PCR. The 5’-fragment (192 bp) covered the first part of the ORF sequence (from +1 to +192 bp) and was amplified from cDNA obtained with the ZmDUR3-specific primer (50 ng as template of PCR reaction). The 3’-fragment (2024 bp) covered most of the remaining ORF sequence (from +172 up to +2196) and was amplified using cDNA obtained with oligo-dT23 (100 ng as template of PCR reaction).
All PCR reactions were performed in a 50μl reaction volume containing 5 × GC Buffer for Phusion® High-Fidelity DNA Polymerase, 0.2 mM ATP, 0.2 mM TTP, 0.3 mM GTP, 0.3 mM CTP, 0.4μM forward primer, 0.4μM reverse primer, 2 U Phusion® High-Fidelity DNA Polymerase (New England Biolabs (UK) Ltd., Hitchin, United Kingdom) following the temperature protocol: 98°C for 30 s; 98°C for 10 s, 58 - 68°C for 30 s, 72°C for 30 s to 2 min, 35°Cycles; 72°C for 10 min. The 5’-fragment was amplified using 5’-CGGAATTCATGGCCGCTGGCGGCGCCGGC-3’ as forward primer and 5’-CAGGAATGAGGTGAAGAGCGCGAAGAAGGCGC-3’ as reverse primer (Tm = 68°C, elongation at 72°C for 30 s). The 3’-fragment was amplified using 5’-TTCTTCGCGCTCTTCACCTC-3’ as forward primer and 5’-CGCGGATCCTTAAGCTAGCGAAAGATTATCTTCATC-3’ as reverse primer (Tm = 58°C, elongation at 72°C for 2 min). The 5’- and 3’-fragments of the ZmDUR3-ORF were assembled using the approach of Assembly PCR. The PCR reaction was carried out with 10 ng 5’-fragment and 10 ng 3’-fragment, as template; using 5’-CGGAATTCATGGCCGCTGGCGGCGCCGGC-3’ as forward primer and 5’-CGCGGATCCTTAAGCTAGCGAAAGATTATCTTCATC-as reverse primer (Tm = 62°C, elongation at 72°C for 1 min 30 s). The full-length ZmDUR3-ORF [GenBank: KJ652242] was amplified and cloned into the S. cerevisiae expression vector pDR197 [21] using the restriction sites for EcoRI and BamHI. The nucleotide sequence was verified by sequencing.
ZmDUR3mod-ORF cloning
In order to reduce the GC content and to facilitate the expression of ZmDUR3 in heterologous organisms, 48 nucleotides in the first 216 nt of ZmDUR3 were modified. These modifications are all synonymous substitutions occurring only at the third base of the codons (the codon-usage preference in yeast was chosen as described by http://www.kazusa.or.jp/codon/). This modified ZmDUR3, called ZmDUR3
mod
[GenBank: KJ652243], differs from the ZmDUR3 only at nucleotide level, while the encoded amino acids remain unchanged (Additional file 5: Figure S5).
The modified region was obtained by assembling two primers, Assembly-1 Primer (5’-GGAATTCATGGCTGCTGGTGGTGCTGGTGCTTGTCCTCCACCAGGTCTAGGTTTTGGTGGTGAATATTATTCTGTTGTTGATGGTGCTTGTAGTCGTGATGG -3’) and Assembly-2 Primer (5’-GGTGCTTGTAGTCGTGATGGTAGCTTTTTTGGCGGTAAACCAGTTCTAGCTCAAGCTGTTGGTTATGCTGTCGTTCTTGGTTTTGGTGCTTTCTTCGCGCTCTTCACCTC-3’), which were synthetized in vitro (Microsynth AG, Balgach, Switzerland).
Two consecutive Assembly PCR reactions were performed to add the long primers to the 3’-fragment.
In the first PCR reaction, 10 ng of 3’-fragment were used as template, while Assembly-2 Primer and 3’-fragment were assembled by PCR, i.e. 10 ng of 3’-fragment were used as template; while Assembly-2 Primer and 5’-CGCGGATCCTTAAGCTAGCGAAAGATTATCTTCATC-3’ were used as forward and reverse primers, respectively (Tm = 62°C elongation at 72°C for 1 min 30 s). 10 ng of purified PCR product were used as template for the consecutive PCR with forward and reverse primers: Assembly-1 Primer and 5’-CGCGGATCCTTAAGCTAGCGAAAGATTATCTTCATC-3’ (Tm = 62°C, elongation at 72°C for 1 min 30 s).
Using the restriction sites EcoRI and BamHI, the full-length ZmDUR3
mod
-ORF was cloned into vector pDR197 [21] and sequenced.
Although the optimization of codon usage in ZmDUR3mod was developed for a better expression in yeast, the modified sequence was also used to perform the functional characterization of DUR3 in tobacco protoplasts and A. thaliana, since also in these latter organisms a high GC content might interfere with the translation of the transcripts.
Expression in Saccharomyces cerevisiae
S. cerevisiae strain YNVWI (Δura3, Δdur3 [13]) was transformed with vector pDR197 (negative control) or plasmids harbouring the ORF sequences (pDR197-ZmDUR3 and pDR197-ZmDUR3
mod
) as described by Liu et al.[13]. Transformants were first selected on synthetic dextrose minimal medium [31] with Oxoid agar (Difco, Detroit, USA) [32]. Single colonies were tested on urea (1, 2 or 3 mM) or ammonium sulphate (0.5% w/v) as sole N source. The pH of the medium was adjusted with 1 m KOH (pH 5.6). The cells were grown for 2–3 days at 28°C.
Protein localization in Nicotiana tabacumprotoplasts
For transient expression of ZmDUR3
mod
in tobacco protoplasts, two plasmids harbouring the sequence for the Green Fluorescent Protein (GFP) were fused at the N- or C-terminus of ZmDUR3 using vectors pUC18-Sp-GFP6 and pUC18-GFP5T-Sp [22]. ZmDUR3
mod
-ORF sequence without stop codon was amplified using primers (5’-ATAACTAGTATGGCTGCTGGTGGTGCTGG-3’, 5’-ATAtAGATCTGCAGCTAGCGAAAGATTATCTTCATCG-3’), and cloned into pUC18-Sp-GFP6 using the SpeI and BglII sites, yielding ZmDUR3
mod
: GFP. On the other hand, to obtain the GFP: ZmDUR3
mod
construct, the ZmDUR3
mod
-ORF sequence with stop codon was amplified using primers (5’-ATATCTAGAATGGCTGCTGGTGGTGCTGG-3’, 5’-ATAATGCATTTAAGCTAGCGAAAGATTATCTTCATCG-3’), and cloned into pUC18-GFP5T-Sp using the NheI and PstI sites.
Protoplast isolation and transformation was performed as described earlier [33]. For co-localization experiments pUC-PTR1-Sp-EYFP [22] was used as marker for the plasma membrane. Tobacco protoplasts were co-transformed with either pUC18-ZmDUR3
mod
-GFP6 or pUC18-GFP5T-ZmDUR3
mod
and pUC-PTR1-Sp-EYFP. As control, free GFP (pUC18-GFP5T-Sp) was transiently expressed in tobacco protoplasts. As reported by Komarova et al.[22], protoplasts were examined with a SP2 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany), excited with an argon laser at 458 nm for GFP and 514 nm for YFP. Fluorescence was detected at 492–511 nm for GFP, at 545–590 nm for YFP and 628–768 nm for chlorophyll epifluorescence detection. Diameter of tobacco protoplasts was approximately 40μM.
Generation of ZmDURmod-overexpressing Arabidopsis lines and growth phenotyping
The ZmDUR3
mod
-ORF was excised from pDR197-ZmDUR3
mod
using EcoRI and BamHI and ligated into vector pBF1 [34] at the EcoRI and BglII sites. Using this pBF1- ZmDUR3
mod
construct as template, the ZmDUR3
mod
-ORF was amplified using primers (5’-ATTTAGGTGACACTATAG-3’, 5’-CGCGGATCCTTAAGCTAGCGAAAGATTATCTTCATC -3’) and cloned into the final vector pCHF5 [35] in the BamHI site, generating a construct named pCHF5-ZmDUR3
mod
. Arabidopsis atdur3-3 plants [18] were transformed by dipping inflorescences into a cell suspension (OD600 = 0.6) of Agrobacterium tumefaciens GV3101 harbouring pCHF5-ZmDUR3
mod
, as described by Clough & Bent [36]. Harvested seeds were germinated on soil; plants at two-leaf-stage were treated with glufosinate (150 mg l-1; BASTA® 200, Bayer CropScience Deutschland GmbH, Langenfeld, Germany) to select transformed lines. The experiments were performed using independent ZmDUR3-overexpressing lines of T2 or T3 generation.
For growth complementation tests, surface-sterilized seeds were grown on agar plates as described by Kojima et al.[18]. Plants were grown on modified half-strength Murashige and Skoog (MS) medium without N, supplemented with 1μM NiCl2 and 50μM KNO3. Either 500μM NH4NO3 or 500, 1000 and 3000μM urea were added as N sources, alternatively no N was added (negative control). Col-0, atdur3-3 and three atdur3-3 transformed lines (atdur3-3 + ZmDUR3-A, -B, -C overexpression lines) were cultured for 16 days in a growth chamber with photoperiod, 24 h; light intensity, 220μMol m-2-s-1; temperature, 20-22°C; relative humidity, 70 to 80%.
Hydroponic culture of Arabidopsis plants and 15[N]-urea root uptake
Arabidopsis thaliana seeds (Col-0; atdur3-3; atdur3-3 + ZmDUR3-A, -B, -C overexpression lines) were germinated on half strength MS-agar medium as described by Norén et al.[37]. After 10 days, the seedlings were transferred for 6 weeks to hydroponic conditions as previously described by Kojima et al.[18]. During the entire growth period N was supplied as 1 mM NH4NO3. 4 days before the experiment, plants were transferred to medium lacking N (no N).
Urea influx measurements into plant roots were conducted after rinsing the roots in 0.5 mM CaSO4 solution for 1 min, followed by incubation for 15 min in nutrient solution containing 100μM of 15[N]-urea (98 atom% 15 N; ISOTEC® Stable Isotopes, Sigma Aldrich, Milano, Italy) as the sole N source. After a final rinse of 1 min in 10 mM non-labelled, ice-cold urea and a second rinse of 1 min in 0.5 mM CaSO4 solution, the Arabidopsis roots were sampled and dried at 40°C and analysed as previously described.
Phylogenetic and statistical analyses
Phylogenetic analyses were conducted using MEGA version 6 software [38]. The tree was constructed by aligning the protein sequences by Clustal-W and the evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown in Figure 2 next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
For the experiments with maize and Arabidopsis plants, three independent experiments were performed using six (if not otherwise specified) plants for each sample; each sample was measured performing three technical replicates. Statistical significance was determined by one-way analysis of variances (ANOVA) using Student-Newman-Keuls test, taking P < 0.05 as significant. Statistical analysis were performed using SigmaPlot Version 12.0 software.