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Supporting information Figs S1-S5. Figure s1 Histochemical assay of root H 2 O 2 All of roots from seedlings grown in MS or containing 20 µM ABA for 12.

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Presentation on theme: "Supporting information Figs S1-S5. Figure s1 Histochemical assay of root H 2 O 2 All of roots from seedlings grown in MS or containing 20 µM ABA for 12."— Presentation transcript:

1 Supporting information Figs S1-S5

2 Figure s1 Histochemical assay of root H 2 O 2 All of roots from seedlings grown in MS or containing 20 µM ABA for 12 days were stained by 3, 5-diaminobenzidine (DAB; Sigma) according to the method described by Du et al. (2008). Pictures were taken by a Leica stereomicroscope. Settings were identical for all the pictures in an experiment. Each experiment was repeated at least 5 times with similar results.

3 Figure s2 Histochemical assay of root O 2־ According to Dunand et al. (2007), the roots from Arabidopsis seedlings grown in MS or containing 20µM ABA for 12 days were stained by a solution of nitroblue tetrazolium (NBT) for 15 min in phosphate buffer (pH 6.1). Pictures were taken by a Leica stereomicroscope. Settings were identical for all the pictures in an experiment. Each experiment was repeated at least 5 times. According to Tsukagoshi et al. (2010), relative staining intensity was analyzed.

4 Figure s3 The transcriptional levels of RBOHD and RBOHF genes The mRNA of RBOHF and RBOHD were examined in Atmpk6-3 and WT root tissue by RT-PCR test. The method is same as stated in this manuscript. The primers are as follows. RBOHD: FP, 5′-TGTTTACCCCGGGAACGTGTTGTCTCTA-3′, RP, 5′-TTGCATCACCTTCCTCGTACACACTCGT-3 ′. RBOHF: FP, 5′-GCTCCGATTTCGCTCAATGCAT-3′, RP, 5′-AGCAGTCGAACCGAATAAGAACC-3 ′.

5 The transcriptional levels of PRX57 and PRX34 genes Analysis in RT-PCR showed the mRNA content of PRX57/34 in Atmpk6 mutant and WT root tissue. The method is same as stated. The primers are as follows. PRX34: FP, 5′-CTGCTTTGTTAATGGTTGTGACGC-3′, RP, 5′-TCGCTCTGGAT AAGACCTTTTCGC-3′; PRX57: FP, 5′- CGACTGTTTCGTTAAGGGCTGTGA-3′; RP: 5’- CAATGGACTCGACTGGTCTAGTGC-3’.

6 ABA-induced H 2 O 2 accumulation in the loss-of-function mutant prx34 (SALK_051769) seedling roots The test method was described in the section of Materials and methods. Figure s4

7 H 2 O 2 -elongated root cell in prx34 seedlings The test method was described in the section of Materials and methods.

8 Figure s5 The transcriptional levels of PM-located Ca 2+ transporters genes Analysis in RT-PCR showed the mRNA content of Ca 2+ transporters, including the glutamate receptors (GLRs) and the cyclic nucleotide gated channels (CNGCs), in Arabidopsis root tissue according to Vadassery et al. (2009). The method is same as stated. The gene-specific primers are as follows.

9 CNGC3 (FP: 5'-GAAGCC CGAGCGATTTTGTC, RP: 5'-GGTTTAAAGCAGCACCAGCC); CNGC10 (FP: 5'-TGTTTAGGTTCA AAGATGAAGGCA, RP: 5‘-ATCGCCAAAGCAACCACAC); CNGC15 (FP: 5'-ACCGGTGTTGTAACCGAGAC, RP: 5'-AGCTGAGGTTCTTCAAGCCC); CNGC20 (FP: 5'-CCTCGAACGCTCTTCTGTAAA, RP: 5'-CTAGTTATAG CCTTTAGTTTGTA); GLR1.3 (FP: 5'-GGCGGGAACTCGTTGTTAGA, RP: 5'-GGACTGTACACGAACACCGT); GLR2.5 (FP:5'-AGGAGGCCATCAGAGA GCTT, RP: 5'- AGCCAAAGCCATCAGCCTTA); GLR3.1 (FP: 5'- AACGTAGTG GCTTCCTCAGC, RP: 5'- CACCACATCTGACCAGCCAT).

10 Analysis in RT-qPCR showed the mRNA content of Ca 2+ transporters, including the glutamate receptors (GLRs) and the cyclic nucleotide gated channels (CNGCs), in Arabidopsis root tissue according to Vadassery et al. (2009). The method is same as stated. The gene-specific primers are as follows.

11 CNGC10 (FP: 5'- CTTGACGCGGTTTGCGATAG, RP: 5‘- GGATCTAATGCCCACGGGAG); CNGC15 (FP: 5'- CGACCCGGTTAACGAAATGC, RP: 5'- GCGTGTGGAAGACGGTAAGA); CNGC20 (FP: 5'- GATGCAATCCGTGAGAGGCT, RP: 5'- CGGGGTTTACAGAAGAGCGT); GLR1.3 (FP: 5'- TCACTAGTTCCAGCCTCCGA, RP: 5'- CCGAAACCGTTGGTGGTAGA); GLR2.5 (FP:5'- AACGGAAAGCTAGAGGCGAC, RP: 5'- CGCAGCTTCTTTGCGTTTGT); GLR3.1 (FP: 5'- CTTGGTGGTGGGTTGCTACT, RP: 5'- GCTGAGGAAGCCACTACGTT).

12 References: Du Y, Wang P, Chen J, Song CP. 2008. Comprehensive functional analysis of the catalase gene family in Arabidopsis thaliana. Journal of Integrative Plant Biology 50: 1318-1326. Dunand C, Crèvecoeur M, Penel C. 2007. Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases. New Phytologist 174: 332-341. Tsukagoshi H, Busch W, Benfey PN. 2010. Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root. Cell 143: 606- 616.


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