Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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26 Transgenic plants were selected on M/S medium supplemented with 50 mg/L hygromycin. Histochemical analysis of the promoter activity of ERF110 A 0.5-kb ERF110 promoter region (–289 to + 226 of ERF110 genomic sequence) was amplified by PCR using primers: 5’-CCC AAG CTT AAA CGA AAG TGA TAA CAT ATA TCA-3’ and 5’-CTA GTC CCA TGG CCG TGC CAA ACC TATT-3’. Following digestion by Kpn I and Nco I, PCR products were inserted into a modified binary vector pCambia1301 (digested by Hind III and Nco I) and fused with a GUS reporter gene. The ProERF110-GUS was introduced into wild-type Col-0 Arabidopsis by floral dip. Transgenic plants were selected on an M/S agar plate with 25 mg/L hygromycin. More than 15 seedlings of the T2 generation propagated from three independent transgenic lines were grown for further analysis. They showed identical GUS stain patterns for each time-point of the treatments. The GUS assay was performed to quantify GUS activity after different treatments on three sets of independent biological samples. The histochemical GUS staining and assay followed previously described method (Wang, N.N. et al., 2005). Immunoprecipitation of the endogenous ERF110 Frozen Arabidopsis plant tissue (1 g) was ground to a fine powder with an ice-cold mortar and pestle. The fine tissue powder was extracted with 3 mL extraction buffer containing 50 mM sodium phosphate pH 7.6, 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 1% glycerol 2-phosphate, 20 mM sodium pyrophosphate, 10 mM sodium vanedate, 10 mM sodium molybdate, 10 mM sodium tartrate, 1 mM PMSF, 0.5% phosphatase inhibitor cocktail 2 (Sigma), 0.5% protease inhibitor (complete EDTA free, Roche) and 2% PVPP. The extract was centrifuged at maximum speed in a bench-top centrifuge for 10 min at 4 °C to remove cell debris. The lysate was then incubated with 60 μL NHS-activated beads that had been conjugated with home-made anti-ERF110 polyclonal antibodies for 2 h at 4 °C. After a brief washing with the extraction buffer for three times, the endogenous ERF110 was eluted with 100 μL SDS-PAGE loading buffer and loaded onto a SDS-PAGE gel to perform western blot analysis. Anti-ERF110 antibodies were raised in rabbits using His6-ERF110 as the antigen. Custom-made monoclonal antibody against the
27 phosphopeptide ARVDpSSHNPIEESM was purchased from GenScript and diluted at a ratio of 1:200 (Kim et al., 2009; Oh et al., 2009). The specificity of this monoclonal antibody was determined using dot blot analysis on 100 ng each of both phosphorylated and non-phosphorylated synthetic peptides. Molecular biological analysis of expression of ERF110 and other flowering-related genes Two-week-old plant tissues were selected for qRT-PCR analysis. RNA samples were extracted by TRIZOL (Invitrogen). qRT-PCR was performed using SuperScript TM III First Strand synthesis kit (Invitrogen). GoTaq qPCR master mix was purchased from Promega for qRT-PCR reaction under the following conditions: 95 °C, 2 min; 60 cycles of (95 °C, 3 s; 59 °C 30 s); 60–95 °C. Gene-specific primers were used. Actin gene was used as an internal control, and sequences of primers were as follows: ERF110-F: 5’-ACG GTG GCG AAT AAA GCA GAA GAG -3’; ERF110-R: 5’-GGC AGA GGT TGT TCC ATT GGT GAA-3’; Actin-F: 5’-GAC CAG CTC TTC CAT CGA GAA-3’; Actin-R: 5’-TCT CGT GGA TTC CAG CAG C-3’; AP1-F: 5’-CAG CAT AAC CAA GGC CAC AA-3’; AP1-R: 5’-CAT TCC TCC TCA TTG CCA TTG-3’.
Page 1 and 2: Plant Physiology Preview. Published
Page 3 and 4: 3 Acknowledgement: This research wo
Page 5 and 6: 5 Introduction Ethylene is known to
Page 7 and 8: 7 whereas ethylene up-regulated ERF
Page 9 and 10: 9 matrix-assisted laser desorption/
Page 11 and 12: 11 To minimize the effect of endoge
Page 13 and 14: 13 Materials and Methods). Western
Page 15 and 16: 15 transcript level in Pro35S-ERF11
Page 17 and 18: 17 Table 2) in the presence of AOA,
Page 19 and 20: 19 role in the regulation of boltin
Page 21 and 22: 21 ethylene-response mutant that ac
Page 23 and 24: 23 sample was set as 1, whereas the
Page 25: 25 C18 Zip-tip column and resuspend
Page 29 and 30: 29 acid-enhanced 1 gene plays a rol
Page 31 and 32: 31 events in plants. Plant Cell 5:
Page 33 and 34: Figure legends 33 Figure 1. Bioinfo
Page 35 and 36: 35 Figure 5. Ethylene represses in-
Page 37 and 38: Table 1. Bolting time of ERF110 WT
Page 39 and 40: Figure 2 A B Anti-ERF110 Anti-Actin
Page 41 and 42: Figure 4 A B C a b Col-0 ctr1-1 erf
Page 43 and 44: Figure 6 A B WT-OX WT-OXA WT-OXD O
Page 45: Figure 8

Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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