2-1. Plant Material and Growth Condition
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type control,
and 19 npf mutants used in nitrate content analysis were obtained from Arabidopsis
Biological Resource Center (ABRC) or The European Arabidopsis Stock Centre (NASC).
AGI code NPF names Published names Mutant line
At3g47960 AtNPF2.10 GTR1 gtr1 (CS879742)
At1g12110 AtNPF6.3 NRT1.1(CHL1) chl1-5
At3g21670 AtNPF6.4 NRT1.3 sper3-3 (SALK_001553)
At1g69870 AtNPF2.13 NRT1.7 nrt1.7-2 (SALK_053264)
At1g22570 AtNPF5.15 npf5.15-1* (CS859376)
At2g26690 AtNPF6.2 NRT1.4 nrt1.4-2 (WiscDsLox322H05)
At5g62680 AtNPF2.11 GTR2 gtr2-1 (Garlic_20_B07)
At1g69850 AtNPF4.6 NRT1.2 ait1-1 (SALK_146143)
At2g02040 AtNPF8.3 PTR2 ptr2-1* (SALK_079073)
At1g62200 AtNPF8.5 PTR6 ptr6-1* (GK-651C03)
At1g68570 AtNPF3.1 npf3.1-1* (SALK_076121)
At3g53960 AtNPF5.7 npf5.7-1* (SALK_068690C)
At3g54140 AtNPF8.1 PTR1 ptr1-1 (SALK_131530)
At2g40460 AtNPF5.1 npf5.1-1* (SALK_000464)
At1g22540 AtNPF5.10 npf5.10-1* (SALK_141062)
At5g13400 AtNPF6.1 npf6.1-1* (SALK 007230)
At5g14940 AtNPF5.8 npf5.8-1* (SALK_039348)
At1g72140 AtNPF5.12 npf5.12-1* (Garlic_168_G10)
At1g72130 AtNPF5.11 npf5.11-1* (SALK_042211)
* The novel mutants, which have not been published, were named here with their NPF names or published name of the gene. Their gene structures were presented in Supplementary Figure 1 to 11, with the primers list in Supplementary Table 1.
Plants were grown hydroponically containing different nitrate concentrations depending
on experimental designs with 1 mM KH2PO4/K2HPO4 and basal nutrient (2 mM MgSO4,
1 mM CaCl2, 0.1 mM FeSO4-EDTA, 50 M H3BO3, 12 M MnSO4．2H2O, 1 M ZnCl2,
1 M CuSO4．5H2O, 0.2 M NaMoO4．2H2O), 0.5g/L MES at pH 5.5 (adjust with KOH)
under long day condition (16h of light/8h of dark cycles; 95-110 mole m-2 s-1 PPDF).
After four days, seeds were germinated on rockwool, and the full nutrient medium was
applied twice a week after germination. The light intensity was measured with a LI-250A
light meter with a LI-190SA quantum sensor (LI-COR).
For primary nitrate response, seeds of Col-0 and mutants were surface-sterilized
with 70% ethanol for 2 minutes then sterilization solution (0.5% SDS and 20% bleach)
for 15 minutes. Approximately 240 seeds were sown on nylon netting supported by
eight-day-old seedlings were transferred to about 57 ml of 12.5 mM ammonium succinate
medium, which the pH was changed to 5.5 by adding HCl, for two treatments, 16 hours
and 3 hours respectively. After the 3-hour treatment, the plants were transferred to
mediums containing 25 mM or 200 M KNO3, pH 5.5, and then the seedlings were
collected at 0, 15, 25, 35, 45, 60, and 120 minutes after transferred. Two vessels were
pooled together for each experiment (approximately 60 seedlings).
Plants for measurement of primary root length were grown on plates. Seeds of wild
type and mutants were surface-sterilized as described previously and sown on 0.2 mM
KNO3, 5 mM KNO3 plates at pH 5.7 and 5 mM ammonium succinate plate at pH 6.5.
After stratification at 4˚C for 3 days, the plates were taken out and put under continuous
light for 4 days, and then the homogeneous seedlings were shifted to new plates with the
same nutrient and pH level for another 6 days. The photos were taken every day at noon,
and the measurement of the length of primary root was using ImageJ software (Schneider
et al., 2012).
2-2. Genomic DNA extraction
7-day-old seedlings was ground with 0.5 mL Urea extraction buffer (7 M Urea, 1%
Sarcosyl, 50 M Tris, pH 8.0, 35 mM NaCl, 20 mM EDTA, pH 8.0) and mixed with 0.4
mL phenol/chloroform/IAA (25:24:1) mixture. Mixed well and centrifuged at 14000 rpm
for 15 minutes, and then the supernatant was transferred to a new microtube, the equal
volume of IPA and 0.1 volume of 3 M sodium acetate and precipitated at 4˚C overnight.
The genomic DNA was pelleted by centrifuged at 14000 rpm for another 15 minutes and
dissolved in water.
2-3. Quantitative PCR analysis
The RNA of samples were extracted with TRIzol reagent (Invitrogen), and then
cDNAs were synthesized using oligo (dT) primers and ImProm-II reverse transcriptase
(Promega). Quantitative PCR was performed with 2x LightCycler 480 SYBR green I
Master Mix (Roche). The initial denaturing step at 95°C for 10 minutes, followed by 40
cycles of 95°C for 10 seconds, 59°C for 5 seconds, and 72°C for 11 seconds. After PCR
cycles, the melting temperature of the PCR product was measured. The gene expression
was analyzed with gene-specific primers and was normalized with UBQ10.
2-4. Nitrate Content Analysis by HPLC
The plant tissue of root or shoot were collected and frozen in liquid nitrogen
immediately, and then samples were dried by lyophilization. To extract nitrate, samples
were boiled in water (1000 L/mg dry weight) for 30 minutes and then freeze-thawed
once. After filtering through 0.2 m polyvinylidene fluoride membrane (Pall
Corporation), nitrate content of samples was determined by HPLC (Thayer and Huffaker,
1980) using a PARTISIL 10 SAX (strong anion exchanger) column (HICHROM) with
50 mM KH2PO4 buffer, pH 3.0, as the mobile phase.
2-5. 15NO3- Labeling Assay
Plants were grown hydroponically 17 days as mentioned above. After the light was
on, plants were transferred to 2 mM KNO3 hydroponic medium containing a 49% excess
of 15N for 5 minutes, washed twice with 0.1 mM CaSO4. The leaves were collected by
order from old to young along with the root in tin capsule, and then dried in 80˚C oven
for two days. 15N abundance in individual leaf was analyzed as described elsewhere using
a continuous-flow isotope ratio mass spectrometer coupled with a carbon nitrogen
elemental analyzer (SERCON)(Fan et al., 2009).
2-6. GUS staining
Transgenic plants were generated by Ya-Yun Wang, with a 2.7-kb genomic
fragment from the promoter to partial second exon (Wang, 2011). Plants were grown on
MS plates (CAISSON) under continuous light. Histochemical staining for GUS activity
was performed on plants at vegetative stage. The whole plant was vacuum infiltrated for
45 minutes at room temperature in 0.5% formaldehyde, 0.05% Triton X-100, 50 mM
sodium phosphate buffer at 7.0. After rinsed three times with 50 mM sodium phosphate
buffer at 7.0, the plants were incubated in staining buffer at 37˚C in dark overnight,
which contains 2 mM X-Glu (Gold BioTechnology, Inc.), 0.5 mM potassium ferricyanide,
0.5 mM potassium ferrocycide, 0.05% Triton X-100, and 50 mM sodium phosphate
buffer at 7.0. The plants were rinsed three times with 50 mM sodium phosphate buffer at
7.0, and then be fixed overnight in 2% formaldehyde, 0.5% glutaraldehyde, and 100 mM
sodium phosphate buffer at 7.0. Pigments were removed by immersing plants in 15%,
30%, 50%, 70% ethanol successively.