Fig. S1

Fig. S1Characterization of npf3.1-2 and npf3.1-3 mutants. Absence of NPF3.1 cDNA full length was verified by RT-PCR using the primers For1 and Rev for npf3.1-3 (b) and For3 and Rev2 for npf3.1-2 (c) as indicated in (a)

Fig. S2


Fig. S2Subcellular localization of a C-terminal fusion NPF3.1-GFP in root cells. Root of seven-days-old T3 plantlets grown on Arabidopsis media (Duchefa) were observed with the confocal microscope SP5 (Leica). From left to right: GFP fluorescence (green), 0.1µg ml-1 FM4-64 (magenta), and merged images of GFP and FM4-64 fluorescence, giving yellow co-localization. Scale bar: 15 µm. The different fluorochromes were detected using laser lines 488 nm (GFP and FM4-64). Each image shown represents a single focal plane

Fig. S3

Fig. S3Influence of exogenous GA3 on germination of npf3.1 mutants. Seeds of Col, npf3.1-2, npf3.1-3 mutants were stratified and grown on 0.5% agarose (control) or 0.5% agarose containing 10 µM paclobutrazol, with or without 1 µM GA3. Germination was determined as cotyledon greening after 5 days. Mean values of three technical replicates are given with standard deviation. Similar results were obtained in three independent experiments using two seed batches. Statistically different mean values were indicated using Tukey range test P=0.05 (one way ANOVA for each condition of germination)

Fig. S4

Fig. S4Influence of exogenous GA3 on hypocotyl elongation of npf3.1 mutants and npf3.1 35S:GFP-NPF3.1 complemented lines grown on 0.25 mM nitrate.For measuring the hypocotyl length, seeds of Col, npf3.1-2,npf3.1-3 mutants and npf3.1 35S:GFP-NPF3.1 complemented lines (B3, J5, L4,G5) were sown on vertical plates containing 0.25mM nitrate and with or without 1 µM GA3. Hypocotyl length was measured at 11 days after germination of light-grown plants with a 16/8 h light photoperiod.Mean values of twenty biological replicates are given with standard deviation. Statistical different mean values were indicated using Tukey range test P=0.05 (one way ANOVA)

Fig.S5

Fig. S5Expression level of NPF3.1 during seed germination of water imbibed seeds, as shown in publicly available transcriptomic data in Arabidopsis eFPbrowser ( (Nakabayashi et al. 2005)

Table S1 Purification by solid phase columns

Column / Steps
WAX (Oasis WAX; Waters) / 1 / Wash the columns successively with bed volume of acetonitrile and metanol and then with half bed volume of 0.1 M KOH
2 / Equilibrate the columns with bed volume of water containing 1 % (v/v) acetic acid
3 / Load the samples that have been dried up and dissolved in water containing 1 % (v/v) acetic acid to the columns
4 / Wash the columns several times with bed volume of water containing1 % (v/v) acetic acid and then with bed volume of acetonitrile
5 / Elute GAs twice with 80 % (v/v) acetonitrile containing 1 % (v/v) acetic acid
SepPak silica (SepPak silica; waters) / 1 / Wash and equilibrate the columns several times with bed volume of chloroform:ethylacate=1:1 (v/v) containing 1 % (v/v) acetic acid
2 / Load the samples that have been dried up and dissolved in chloroform:ethylacetate=1:1 (v/v) containing 1 % (v/v) acetic acid
3 / Collect flow through containing GAs after the step 2
4 / Further elute GAs twice with bed volume of chloroform:ethylacate=1:1 (v/v) containing 1 % (v/v) acetic acid

Table S2 Conditions of LC

Solvent A / Solvent B / Gradient (composition of solvent B) / column
Water containing 0.01 % (v/v) acetic acid / MeCN containing 0.05 % (v/v) acetic acid / Constant at 3 % linear for 0.5 min / ACQUITY UPLC BEH phenyl column (Waters, 17 μm, 2.1 × 50 mm)
Linear gradient from 3 to 20 % over 2.5 min
Linear gradient from 20 to 40 % over 5 min
Constant at 40 % for 2 min

Table S3 Parameters of tandem mass spectrometer

Compound / Retention time on LC (min) / Polarity of ESI / IonSpray vortage (kV) / Desolvation temperature (°C) / Declustering potential (V) / Collision energy (V) / Precursor ion (m/z) / Scan range (m/z) / Qualifier ion (m/z)
D2-GA8 / 2.3 / - / -4.0 / 600 / -90 / -30 / 365.2 / 100-1000 / 277.2
GA8 / - / -4.0 / 600 / -90 / -30 / 363.2 / 100-1000 / 275.2
D2-GA1 / 3.3 / - / -4.0 / 600 / -90 / -40 / 349.2 / 100-1000 / 275.2
GA1 / - / -4.0 / 600 / -90 / -40 / 347.2 / 100-1000 / 273.2
D2-GA3 / 3.3 / - / -4.0 / 600 / -90 / -40 / 347.2 / 100-1000 / 223.2
GA3 / - / -4.0 / 600 / -90 / -40 / 345.2 / 100-1000 / 221.2
D2-GA19 / 4.6 / - / -4.0 / 600 / -80 / -45 / 363.2 / 100-1000 / 275.2
GA19 / - / -4.0 / 600 / -80 / -45 / 361.2 / 100-1000 / 273.2
D2-GA20 / 4.8 / - / -4.0 / 600 / -90 / -35 / 333.2 / 100-1000 / 289.2
GA20 / - / -4.0 / 600 / -90 / -35 / 331.2 / 100-1000 / 287.2
D2-GA44 / 5.0 / - / -4.0 / 600 / -90 / -40 / 347.2 / 100-1000 / 303.2
GA44 / - / -4.0 / 600 / -90 / -40 / 345.2 / 100-1000 / 301.2
D2-GA53 / 5.7 / - / -4.0 / 600 / -90 / -40 / 349.2 / 100-1000 / 305.2
GA53 / - / -4.0 / 600 / -90 / -40 / 347.2 / 100-1000 / 303.2
D2-GA4 / 6.6 / - / -4.0 / 600 / -90 / -30 / 333.2 / 100-1000 / 259.2
GA4 / - / -4.0 / 600 / -90 / -30 / 331.2 / 100-1000 / 257.2
D2-GA24 / 6.8 / - / -4.0 / 600 / -90 / -40 / 347.2 / 100-1000 / 259.2
GA24 / - / -4.0 / 600 / -90 / -40 / 345.2 / 100-1000 / 257.2
D2-GA15 / 8.0 / - / -4.0 / 600 / -90 / -40 / 331.2 / 100-1000 / 259.2
GA15 / - / -4.0 / 600 / -90 / -40 / 329.2 / 100-1000 / 257.2
D2-GA9 / 8.0 / - / -4.0 / 600 / -90 / -35 / 317.2 / 100-1000 / 273.2
GA9 / - / -4.0 / 600 / -90 / -35 / 315.2 / 100-1000 / 271.2
D2-GA12 / 8.7 / - / -4.0 / 600 / -90 / -40 / 333.2 / 100-1000 / 315.2
GA12 / - / -4.0 / 600 / -90 / -40 / 331.2 / 100-1000 / 313.2