Supporting information Figs S1-S5

Figure s1

Histochemical assay of root H2O2

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.

Figure s2

Histochemical assay of root O2־•

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.

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′.

The transcriptional levels of PRX57 and PRX34 genesAnalysis 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’.

ABA-induced H2O2 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

H2O2-elongated root cell in prx34 seedlings

The test method was described in the section of Materials and methods.

Figure s5

The transcriptional levels of PM-located Ca2+ transporters genesAnalysis in RT-PCR showed the mRNA content of Ca2+ 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.

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).

Analysis in RT-qPCR showed the mRNA content of Ca2+ 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.

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).

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.