Supplemental data

Supplemental Fig. S1 Effect of MdCIPK6L expression on salt tolerance in WT, sos2 mutant and transgenic lines Arabidopsis. Both MdCIPK6L and MdCIPK6LT175D complemented the function of Arabidopsis sos2 mutant which was supersensitive to high salinity. Transgenic lines (cMdCIPK6L-1, -3, -4 and cMdCIPK6LT175D-1, -2, -3), sos2 mutant and wild-type (WT) under MS medium contain 0, 100 or 200 mM NaCl for 10 days.

Supplemental Fig. S2 Tolerance of MdCIPK6L transgenic Arabidopsis lines to salt, PEG-induced osmotic and chilling stresses.

Supplemental Fig. S3 Effects of MdCIPK6L expression on the tolerance to salt, PEG-induced osmotic and chilling stresses in transgenic apple. a Na+ content in leaves of WT and transgenic apple lines in Fig. 5b. b Relative water content (RWC) of WT and transgenic lines in Fig. 5c. c Electrolyte leakage of leaf cells in WT and transgenic lines in Fig. 5d. Each value is the mean ± SE of three independent determinations, and different letters indicates significant differences at P < 0.05 (Student’s t-test) when comparing values within the same group of values.

Supplemental Fig. S4 Effects of MdCIPK6L overexpression and suppression on the tolerance to salt, drought and chilling stresses in self-rooted transgenic apple plantlets. a Salt tolerance of MdCIPK6L overexpression and antisense suppression lines. Plant phenotypes of WT and transgenic lines at day 12 after exposed to 200 mM NaCl. b Drought tolerance of MdCIPK6L overexpression and antisense suppression lines. Plant phenotypes of growth recovery of WT and transgenic lines at day 6 after rewatering, following water withdraw for 15 days. c Chilling tolerance of MdCIPK6L overexpression and antisense suppression transgenic lines. Plant phenotypes of growth recovery of WT and transgenic lines at day 10 after transferring to normal conditions, following 4 °C chilling for days. d Root systems of WT and transgenic apple self-rooted plantlets.

Supplemental Table S1 Primers for gene cloning, point mutagenesis and vector construction.

Constructions / Primers
degenerate primer / forward: 5’-TTTWCNGAGCACYYNMRNCAG-3’
reverse: 5’-GACMTRGAGATTATGTGAAAC-3’
5’RACE / 5GSP1: 5’- CGCCTTGGCTCCGTCGT-3’
5GSP2: 5’- ATGACCTCCGGAGCCACGT-3’
3’RACE / 3GSP1: 5’-AGTCGTACTGACGAAACAGGAGC-3’
3GSP2: 5’-AAGATCACGGCGAGGCAGAC-3’
MdCIPKL / forward: 5’-ATGGCCGAGCAGAAAG-3’
reverse: 5’-AATCTTAATCAATCAAGCC-3’
MdCIPKLT175D / Forward1: 5’-ATGGCCGAGCAGAAAG-3’
Reverse1: 5’-GCATGTGTCATGGAGGAGCCCGT-3’
Forward2: 5’-ACGGGCTCCTCCATGACACATGC-3’
Reverse2: 5’-AATCTTAATCAATCAAGCC-3’
antiCIPKL / forward: 5’-GCTCGTTGCTGCATGGCAAG-3’
reverse: 5’-CACCTTCAGCTTGCTGAGACA-3’

Supplemental Table S2 Primers for expression analysis using semi-quantitative RT-PCR.

Gene name / Primers
MdCIPKL / forward: 5’-GATAAAGAAGAGCGACTCGATGG-3’
reverse: 5’-CTTAATCAATCAAGCCGCCGT-3’
18s rRNA / forward: 5’-GGGTTCGATTCCGGAGAGG-3’
reverse: 5’- CCGTGTCAGGATTGGGTAAT-3’
AtSOS1 / forward: 5’-ATACTCGTATGGTCTGGTTTGAGG-3’
reverse: 5’-TCATCAAGCATCTCCCAGTAAG-3’
AtSOS2 / forward: 5’-ATGACAAAGAAAATGAGAAGAGTG-3’
reverse: 5’-TCAAGCCTCCCTTTATGAAC-3’
AtACTIN / forward: 5’-GGAAAGGATCTGTACGGTAAC-3’
reverse: 5’-TGTGAACGATTCCTGGAC-3’
SlACTIN / forward: 5’-CTTCAGTCCACAATCGGTGG-3’
reverse: 5’-CATTCCGAGTTGAGCTGCTG-3’

Supplemental Table S3 Primers for yeast two-hybrid assays.

Constructions / Primers
pGBKT7-MdCIPKL / forward: 5’-ATGGCCGAGCAGAAAG-3’
reverse: 5’-AATCTTAATCAATCAAGCC-3’
pGADT7-AtCBL3 / forward: 5’-ATGTCGCAGTGCATAGACGG-3’
reverse: 5’-TCAGGTATCTTCCACCTGCGAG-3’
pGADT7-AtCBL4 / forward: 5’-atgggctgctctgtatcgaag-3’
reverse: 5’-ggaagatacgttttgcaattcc-3’
pGBKT7-AtSOS2 / forward: 5’-ATGACAAAGAAAATGAGAAGAGTG-3’
reverse: 5’-TCAAAACGTGATTGTTCTGAG-3’