Supplementary Materials: 1. Supplementary Figures S1-S9. 2. Supplementary Tables S1-S2

Transcription

1 Supplementary Materials: 1. Supplementary Figures S1-S9 2. Supplementary Tables S1-S2 S1

2 1. Supplementary Figures Fig. S1. Genotypes of tcp20 mutants. Fig. S2. Root phenotypes of tcp20 mutants. Fig. S3. Analysis of the tcp20-2cm complemented line. Fig. S4. Pavement cell areas of tcp20 mutants and complemented line. Fig. S5. Root phenotypes of WT and tcp20 mutant plants grown on split-root plates with 1 mm ammonium. Fig. S6. Root phenotypes of chl1 and nlp7 single mutants and double mutants grown on splitroot plates. Fig. S7. Illustration of experiment designed for testing systemic signaling. Fig. S8. Analysis of NRT, NIA1 and NiR mrna levels in WT and tcp20 mutants on uniform plates. Fig. S9. Analysis of NRT, NIA1 and NiR mrna levels in WT and tcp20 mutants on split-root plates. S2

3 Fig. S1. Genotypes of tcp20 mutants. (a) T-DNA insertions in tcp20-2 ( SALK_088460C) and tcp20-4 (SALK_041906C) mutants. (b) Steady-state levels of TCP20 mrna in tcp20 mutants were determined by quantitative RT-PCR using primers upstream (5 ) of the T-DNA insertion. Error bars represent the standard deviation (SD) for three biological replicates of three plants each. S3

4 Fig. S2. Root phenotypes of tcp20 mutants. Seedlings were grown on agarose plates with 5 mm NO 3 - as sole N source. (a) Primary root length for WT, tcp20-2 and tcp20-4 mutant plants at 8 days. (b-c) Total lateral root length (b) and numbers (c) for WT, tcp20-2 and tcp20-4 mutant plants at 10 days. Error bars represent standard errors (SEM) for plants; no statistically significant differences were found. S4

5 Fig. S3. Analysis of the tcp20-2cm complemented line. The tcp20-2 mutant was transformed with 2X35S:3FLAG-TCP20 construct and then analyzed. (A) Time course of second-order LR growth in WT and tcp20-2cm plants on split-root plates with HN (5 mm NO 3 - ) and LN (0 mm NO 3 - ) media. Errors bars represent SEM (n=6-8). (B) The ratio of total second-order LR length in HN versus LN side at 10 days. Errors bars represent SEM (n=6-8). (C) Root phenotypes of tcp20-2cm plant on split-root plates at 10 days are shown. (D) TCP20 mrna in wildtype and tcp20-2cm lines were determined by quantitative RT-PCR. Each bar presents 3 biological replicates with standard deviations. S5

6 Fig. S4. Pavement cell areas of tcp20 mutants and complemented line (tcp20-2cm). True leaves of 10-day-old Arabidopsis were stained with propidium iodide solution (1mg/ml) and then micrographs were taken by confocal microscropy. The pictures were taken in the center of leaves away from margins and midribs. Cell size was measure by using IMAGE J software. Cell area is given in arbitrary units with WT as 100%. Error bars represent SEM for pictures from 6 plants. Asterisks indicate statistical significance relative to WT (P 0.05; t test). S6

7 Fig. S5. Root phenotypes of WT and tcp20 mutant plants grown on split-root plates with HN (5 mm NO 3 - ) and LN (0 mm NO 3 - ) media at 10 days in the presence of 1 mm ammonium. S7

8 Fig. S6. Root phenotypes of chl1 and nlp7 single mutants and double mutants (chl1xtcp20-2 and nlp7xtcp20-2) grown on split-root plates with HN (5 mm NO 3 - ) and LN (0 mm NO 3 - ) media at 10 days. S8

9 Fig. S7. Illustration of experiment designed for testing systemic signaling (A) with photographs of WT root phenotypes in this experiment (B). Arabidopsis roots grown in three different nitrate conditions. C.KNO 3 plants treated both sides with 5 mm KNO 3, C. KCl treated both sides with 5 mm KCl, and Sp.KNO 3 and Sp.KCl plants treated with 5 mm KNO 3 on one side and 5 mm KCl on the other. Plants were grown in a growth chamber at C. S9

10 Fig. S8. Analysis of NRT, NIA1 and NiR mrna levels in WT and tcp20 mutants on uniform plates. Relative mrna levels at 10 days in total roots on 5 mm KNO 3 uniform plates were determined by qpcr. Error bars represent SD (n = 3 biological replicates). S10

11 Fig. S9. Analysis of NRT, NIA1 and NiR mrna levels in WT and tcp20 mutants on split-root plates. Relative mrna levels at 10 days in total roots on LN (0 mm KNO 3 ) or HN (5 mm KNO 3 ) medium were determined by qpcr. Error bars represent SD (n = 3 biological replicates). 2. Supplementary Tables. S11

12 Table S1. Genes that showed evidence of TCP20 binding (compiled from (8, 9)) and evidence of nitrate induction or repression in roots or shoots (10) are shown. Nitrate induction/repression values for each gene are shown as shoot or root ratios. TAIR ID Shoot Root Ratio Ratio From Herve et. al. Plant Physiology (2009) (8) At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g At1g S12

13 At1g At1g At1g At1g At1g At1g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At2g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g At3g S13

14 At3g At3g At3g At3g At3g At3g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At4g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g At5g S14

15 At5g At5g At5g From Danisman et. al. Plant Physiology (2012) (9) AT1G AT1G AT1G AT2G AT2G AT2G AT4G AT4G AT5G AT5G Not Detected Table S2. Primers used in qpcr. S15

16 Gene Forward primer Reverse primer NIA1 GGTGCTGGTGTTTCTGGTCACT GGGTCTGGTCGGGTGTTC NRT1.1 AAAGCTGCCACACACTGAAC ATTGTGCGACTGATAATGTCGT NRT2.1 CCACAGATCCAGTGAAAGG CATTGTTGGGTGTGTTCTC NiR CCGGTAGCCAGTTCTGCG CCTATTCGTCCCCCGACGT TCP20-5 GGAGCTGGTTAGAATTGGG GCCTTGAGAATCATCTTTCTCA NIA1-C1 TATCTCGGATCTCTGCTCTG TATACCGCCCTGAATGAC NIA1-C2 CGGTCACATCTATGACTG GATGAGTTCACCGATACG NIA1-T GCGGGTCCATACGTTCTTACAC TTGGAGGCTAAGTGGCGCATGG NRT2.1-C1 AATTCGGCAAACCACGATGCAG NRT2.1-C2 CAATTGTGCCCTTTGTCTCC ACACAGCTTGCCTATGTACG GTAACGTGCAAGCGACTATC NRT2.1-T GGTAGAGCGGTCATTTGGAAG TAGGGTTCCTAGCCAGTGTTG NRT1.1-U1 AGACAGGACAAACGACTC NRT1.1-U2 ACGTGGTCCTACCAACTATC NRT1.1-U3 CCATTGAAGTTGCGAGAC AATGCGAAGAGGGGAATCG TAAACACATCCTCCGAAAAG GCTTCTTAGACGCCACTA NRT1.1-U4 GCCTCGACAATACTACAAAATCTC ACCAGTTTTAGCCACCACCT NRT1.1-U5 CCACAGTTCCAAAGGTAGAG NRT1.1-U6 AGGCAGCAATAAGGGATCAG ACTGGGAAACTAGCCAAG GTCCAATGTCTCGGATTGTG UBQ5 GAGGCACATACTGTCTAGACAAAC CTAGCTTGTGTGAGACCTAGCC S16