Kim et al. 2011 RAP2.11 modulates AtHAK5 gene expression Supplemental Data p. 1
Supplemental Figure 1. The 42 mutants showed high endogenous AtHAK5 gene expression. RT-PCR analysis of AtHAK5 expression was performed with β-tubulin gene (At5g62690) specific primers. Amplification was performed as follows: 30 s at 94℃, 30 s at 60℃,and 45 s at 72℃for 30 cycles, followed by a 10 min extension at 72℃. Blue boxes indicate negative control (NC), plants transformed with AtHAK5pro:LUC. Red boxes indicate mutants showed high endogenous AtHAK5 gene expression than NC.
Supplemental Figure 2. Analysis of AtHAK5gene expression and plant growth under K+ sufficient and K+deprivedconditions with or without sucrose.
(A)Quantitative Real-time PCR analysis of AtHAK5transcript levels in wildtype Col-0. Two experiments were performed. In the first one (first and second bar), 4-d-old seedling were transferred to either potassiumsufficient or potassium deficient with 2% sucrosemedium. In the second experiment, 4-d-old seedlings weretransferred to either potassiumsufficient or potassium deficient without sucrose medium. AtHAK5 transcript levels were measured in the rootsseven days after transfer.Data represent the mean of three biological replicates.Different letters indicates a significant difference between means at P <0.05 (TUKEY HSD) (n≥50 plants).
(B) 4-d-old seedlings were transferred to potassiumsufficient or potassium deficient with/without sucrose medium. The primary root lengths were measured 0, 3, 5, 7, 10 days after. Data represent the mean ± standard deviation.
Supplemental Figure 3.Root growthunder K+ sufficient and K+deprivedconditions.
(A) Primary root length of plants were grown under complete nutrient conditions for 4 d and then transferred to either full nutrient (+K) or no potassium (-K) for 0, 3, 5, 7, 10 days. Data represent the mean ± standard deviation(n≥50 plants).
(B) Primary root length of plants grown without K+ displayedas a ratio of the primary root length of full nutrient (+K) plants. Data represent the mean ± standard deviation(n≥50 plants).
Supplemental Table 1. Segregation ratios of the F2 progeny from 8-1-23 mutant.
Luciferase activity: No luciferase activity / Approximate segregation ratios / Mutant loci / Χ2223: 76 / 3:1 / 1 / 0.03
F2 progenywere grown on K+ sufficient medium containing 1.75 mM KCl for 6 days. Plants were then sprayed with luciferin and the ratio of plants that showed luciferase activity compared with no luciferase activity was determined. The F2 progeny from a single mutant insertion would result in a 3:1 segregation ratio. The number of mutant loci predicted from the 3:1 segregation ratio and their probability from the chi-square test are shown.
Supplemental Table 2. Microarray analysis of differentially expressed genes between RAP2.11-ox and WT in root. Differential expression determined by FDR P-value < 0.05. Arabidopsis gene locus, Gene product, and annotation from TAIR database. Linear fold change and FDR P-values from ANOVA analysis (see Methods).
Supplemental Table 3. Motif Analysis in Promoter/Upstream Sequences of up- or down-regulated in the RAP2.11-ox.
Group / Motif / # in Querya / % in Queryb / # in Genomec / % in Genomed / % Differencese / P-Valuef / Corresponding geneUp- regulated in root / GCCGGC / 67 / 16.3
(67/411) / 1141 / 3.4
(1141/33518) / 12.9 / 3.65e-26 / AT1G06000 AT1G07610 AT1G07650 AT1G08720 AT1G17860 AT1G21100 AT1G25375 AT1G33080 AT1G33700 AT1G47980 AT1G64210 AT1G64740
AT1G74770 AT1G74780 AT1G75010 AT1G77660 AT2G26420 AT2G29440 AT2G30360 AT2G36590 AT2G37430 AT2G42850 AT2G43590 AT2G44770
AT2G47710 AT3G09240 AT3G13700 AT3G13710 AT3G17780 AT3G22540 AT3G23490 AT3G23550 AT3G49620 AT3G51550 AT3G54070 AT3G54820
AT3G61250 AT4G01420 AT4G09690 AT4G13420 AT4G15210 AT4G16260 AT4G20780 AT4G34270 AT4G36520 AT4G37530 AT4G38410 AT5G01060
AT5G02650 AT5G04850 AT5G08010 AT5G10100 AT5G18840 AT5G20050 AT5G22550 AT5G24490 AT5G25140 AT5G38240 AT5G41680 AT5G46730
AT5G47650 AT5G48070 AT5G54040 AT5G58010 AT5G58750 AT5G64850
AT5G65270
Down-regulated in root / GCCGGC / 6 / 1.7
(6/355) / 1141 / 3.4
(1141/33518) / -1.7 / Not significantly enriched / AT4G14900 AT1G01500 AT1G55675 AT5G04600 AT3G61630 AT3G63360
Up- regulated in leaf / GCCGGC / 5 / 38
(5/13) / 1141 / 3.4
(1141/33518) / 34.6 / 4.46e-05 / AT3G49620 AT2G36590 AT2G30360
AT5G63970
AT4G16515
a Absolute gene number of the motif in query set
b Percentage of sequences in query set containing motif
c Absolute gene number of the motif in genomic set
d Percentage of sequences (out of 33518 in genomic set) containing motif
e (Percentage of sequences in query set) - (Percentage of sequences in genomic set)
f p-value from binomial distribution
Supplementary Table 4. Primers for AtHAK5 promoter deletion fragments.
Number of fragment / Forward primer and reverse primer∆ 1 / 5’- AAGGTACCGGTATGACCAGCACATGTAG-3’
5’-AACTCGAGTACCAGTTAAAACATAGAAT-3’
∆ 2 / 5’-AAGGTACCTTAACTGGATGAATTTTCCA-3’
5’-AACTCGAGAAGGGTGGATAACGGATAAT-3’
∆ 3 / 5’-AAGGTACCATCCACCCTTACCAATATTG-3’
5’-AACTCGAGTAAAAATGTATCCTGAATAT-3’
∆ 4 / 5’-AAGGTACCTACATTTTTATTTTTGAGAA-3’
5’-AACTCGAGTTTATACAAAAAAGTTTGGT-3’
∆ 5 / 5’-AAGGTACCTTTGTATAAAAATATTTGTT-3’
5’-AACTCGAGAGATATCCTGAAGAATTCTA-3’
∆ 6 / 5’-AAGGTACCCAGGATATCTACTTCATAAA-3’
5’-AACTCGAGAGAGTAGGAATATTCATTGG-3’
∆ 7 / 5’-AAGGTACCTTCCTACTCTTTAGCTAGTT-3’
5’-AACTCGAGGCGTCTCAAACACGTATTGC-3’
∆ 8 / 5’-AAGGTACCTTTGAGACGCGTCCCTACCA-3’
5’-AACTCGAGGTGGCTTTTATAGGCGTAAT-3’
∆ 9 / 5’-AAGGTACCTAAAAGCCACTGGCAAAAGA-3’
5’-AACTCGAGTTTTTTTTTTTTTTTTTTTTGT-3’