Supplemental Data. Na Xu et al. (2016). Plant Cell /tpc

Transcription

1 Supplemental Figure 1. The weak fluorescence phenotype is not caused by the mutation in At3g (A) A mutation mapped to the gene At3g Map-based cloning strategy was used to map the mutated site in Mut75 and a candidate gene At3g60240 with a single base mutation was isolated, black arrow indicates the mutation site. Exons and introns are represented by black boxes and lines, respectively, untranslated regions are represented by white boxes. The location of the T-DNA insertion line SALK_ is indicated with triangle. (B) RT-PCR analysis of At3g60240 mrna levels in WT and SALK_ mutant. Total RNA isolated from 7-day-old seedlings grown on ammonium nitrate was analyzed by RT-PCR and a program based on 25 cycles of PCR amplifications was carried out to test the expression of At3g TUB2 serves as a control to show the equal amount of cdna in each reaction. (C) Root fluorescence of F 1 (SALK_ x Mut75) plants. Fluorescence and light images of 4-day-old seedlings grown on nitrate media were captured with a fluorescence microscope. (D) Nitrate induction of gene expression in At3g60240 mutant (SALK_002002) and WT. The seedlings were grown and treated as described in Figure 3 A. The transcripts of the nitrate responsive genes in roots were determined by qpcr. Error bars represent SD of biological replicates (n = 4). 1

2 Supplemental Figure 2. Nitrate induction of the endogenous genes tested is inhibited in the nrg2 mutants, and the expression of NRG2 is not regulated by nitrate, ammonium, and nitrogen-starvation treatments. (A) and (B) Nitrate induction of endogenous genes without nitrogen starvation using ACTIN2 (A) and UBQ10 (B) as reference genes. Seedlings were treated and data were analyzed as described in Figure 3 A. (C) The expression of endogenous genes without treatment. WT and nrg2 plants were grown on medium with 2.5 mm ammonium succinate as the sole nitrogen source for 7 d. Roots were collected for RNA extraction. The transcripts of nitrate responsive genes were determined by qpcr. Error bars represent SD (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the WT (t test). (D) The expression of endogenous genes after KCl treatment. Plants were grown on the medium as used in (C) for 7 d and then treated with 10 mm KCl for 2 h. RNA extraction, gene expression test, and data analysis were performed as described in (C). (E) The expression of endogenous genes after KNO 3 treatment. Plants were grown on the medium as used in (C) for 7 d and then treated with 10 mm KNO 3 for 2 h. RNA extraction, gene expression test, and data analysis were performed as described in (C). (F) Nitrate induction of endogenous genes in plants after treated with low nitrate concentration. The same experiments were performed as described in Figure 3 A, except the treatments with 0.1 mm KNO 3 or KCl as a control instead of the treatments with 10 mm KNO 3 or KCl. (G) Time course of NRG2 expression after different nitrogen treatments. For NH + 4 treatment, seedlings were grown on medium with 10 mm KNO 3 for 7 d, and then treated with 10 mm NH 4 NO 3 for different times. For NO - 3 treatment, seedlings were grown on 2.5 mm ammonium succinate for 7 d, and then treated with 10 mm NH 4 NO 3 for different times. For nitrogen deprivation treatment, seedlings were grown on 10 mm NH 4 NO 3 for 5 d, and then transferred to nitrogen-free medium for different times. RNA extraction, gene expression test, and data analysis were performed as described in (C), except that the seedlings without treatment were used as a control. 2

3 Supplemental Data. Na Xu et al. (2016). Plant Cell /tpc Supplemental Figure 3. Sequence alignment of 15-member, Arabidopsis gene family containing NRG2. The alignment was performed using ClustalX program. The DUF630 and DUF632 domains are indicated by red bars over the sequence. 3

4 Supplemental Figure 4. Morphological phenotype of the nrg2 mutant under different concentrations of nitrate. Seedlings were grown on vermiculite under long-day condition (16 h light/8 h dark) and watered with 10 mm (Figure S4 A and S4 B) and 0.1 mm (Figure S4 C) KNO 3 media, respectively every 3 d. Pictures were taken after 18 d (Figure S4 A) and 30 d (Figure S4 B and S4 C), respectively. 4

5 Supplemental Figure 5. The expression of additional nitrate transport genes in roots is not affected in nrg2 mutants. WT and nrg2 mutant plants (nrg2-1 and nrg2-2) were grown on ammonium nitrate medium for 7 days, and then roots were collected separately for RNA extraction. The relative expression of NRT1.2 (A), NRT1.4 (B), NRT1.5 (C), NRT1.6 (D), NRT1.7 (E), NRT1.8 (F), NRT1.9 (G), NRT1.11 (H), NRT1.12 (I), NRT2.1 (J), and NRT2.6 (K), NRT2.7 (L) was determined using qpcr. Error bars represent SD of biological replicates (n = 4). 5

6 Supplemental Figure 6. The expression of additional nitrate transport genes in leaves is not affected by disruption of NRG2 gene. WT and nrg2 mutant plants were grown on ammonium nitrate medium for 7 days, and then leaves were collected separately for RNA extraction. The relative expression of NRT1.2 (A), NRT1.4 (B), NRT1.5 (C), NRT1.6 (D), NRT1.7 (E), NRT1.9 (F), NRT1.11 (G), NRT1.12 (H), NRT2.1 (I), NRT2.6 (J), NRT2.7 (K) in nrg2 mutants was determined using qpcr. Error bars represent SD of biological replicates (n = 4). 6

7 Supplemental Figure 7. The expression of nitrate reduction genes in nrg2 mutants is not altered compared with that in WT. WT and nrg2 mutant plants were grown on ammonium nitrate medium for 7 days and whole seedlings were collected for RNA extraction. The expression of NIA1 (A), NIA2 (B), NiR (C), GLN1.1 (D), and GLN1.3 (E) were determined by qpcr. Error bars represent SD of biological replicates (n = 4). 7

8 Supplemental Figure 8. The expression of additional nitrate regulatory genes tested is not altered in nrg2 mutants compared with that in WT. WT and nrg2 mutant plants were grown on NH 4 NO 3 and KNO 3 media, respectively, and 7-day-old seedlings were collected for RNA extraction. The expression of NLP7 (A), CIPK8 (B), CIPK23 (C), LBD37 (D), LBD38 (E), LBD39 (F) in WT and two nrg2 mutant plants were detected using qpcr. Error bars represent SD of biological replicates (n = 4). 8

9 Supplemental Figure 9. The expression of NRG2 is not changed in characterized nitrate regulatory gene mutants tested compared with that in WT. WT, nlp7-4, cipk8-1, and cipk23-3 mutant plants were grown on NH 4 NO 3 and KNO 3 media, respectively, and 7-day-old seedlings were collected for RNA extraction. The expression of NRG2 was inspected using qpcr. Error bars represent SD of biological replicates (n = 4). 9

10 Supplemental Figure 10. The expression of NRT1.1 in WT, nrg2 mutants, and complementary lines. (A) The expression of NRT1.1 in nrg2 mutants was decreased. WT and nrg2 mutant plants were grown on media with ammonium succinate for 7 days, and then whole seedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the WT (t test). (B) Time course of NRT1.1 expression in WT and the nrg2 mutants after nitrogen deprivation treatment. Seedlings were grown on 10 mm NH 4 NO 3 for 5-7 days, and then transferred to nitrogen-free medium for different times. Roots were collected for RNA extraction and the data were analyzed using the seedlings at time 0 h of treatment as a control. Error bars represent SD of biological replicates (n = 4). (C) The expression of NRT1.1 was overexpressed in NRT1.1/nrg2-2 line. WT, nrg2-2 and NRT1.1/nrg2-2 plants were grown on ammonium nitrate medium for 7 days and whole seedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4). 10

11 Supplemental Figure 11. BiFC assay revealed direct interaction between NRG2 and NLP7 when plants were treated with nitrate after nitrogen starvation. N- and C-terminal fragments of yellow fluorescence protein (YFP N and YFP C ) were fused to NRG2 and NLP7, respectively. N. benthamiana plants were grown on ammonium nitrate medium for 3 weeks followed by 2 weeks of nitrogen starvation. Different combinations of expression vectors encoding NRG2-YFP N and NLP7-YFP C and controls (indicated on the left of the panel) were transiently transformed into leaves and then watered with ammonium nitrate for 3-4 days. Presence of YFP signal indicates reconstitution of YFP through protein interaction of the tested pairs. N. benthamiana cells showing YFP fluorescence in the nucleus were observed and marked by red arrows. Bar = 5 μm. 11