Plant materials and growth conditions
Col-0 was used as the wild type unless stated otherwise. ein2-1, etr1-3, ate1-2;ate2-1, prt6-5, proWOX4-erYFP, proPXY:GUS, proPXY:erVenus and 35S:EIN3-GFP have been described previously10,13,27,37,38,39,40. ate1-2;ate2-1 mutants were reciprocally backcrossed into Col-0 for three generations and transfer DNA insertions were confirmed by genotyping to obtain double-homozygous mutants.
Seeds were sown on half-strength MS (Duchefa) plates supplemented with 0.05% MES (Duchefa), 1% agar (Duchefa) and 1% sucrose (Duchefa), pH 5.8. MS medium also contained vitamins for all experiments except for ethylene measurement from roots. After incubation at 4 °C for more than 2 days, the plates were moved to the growth chamber (22 °C; 16 h light, 8 h dark). The day that the plates were placed in the growth chamber is defined as day 0. The seedlings were moved to the new MS agar plate around day 7; for the experiment in shoots, the seedlings were transferred to soil 1 or 2 weeks after germination and were grown in a greenhouse (16 h light, 8 h dark) unless stated otherwise. For examining normal periderm, we used 14- or 16-day-old seedlings as periderm is established at that age. For assessing regeneration, we usually used 17- to 21-day-old seedlings.
The Arabidopsis Genome Initiative locus codes for the genes are as follows: ATE1, AT5G05700; ATE2, AT3G11240; EBF1, AT2G25490; EIN2, AT5G03280; EIN3, AT3G20770; ETR1, AT1G66340; PBP1, AT3G16420; PCO1, AT5G15120; PCO2, AT5G39890; PER15, AT2G18150; PER49, AT4G36430; PRT6, AT5G02310; PXY, AT5G61480; RPS5A, AT3G11940; WOX4, AT1G46480.
Cloning
From the Col-0 genome, the 643-base-pair (bp) EBF1 3′ UTR with its downstream 72-bp sequence (from +2780 to +3495) was amplified by PCR using H122_EBF1_URT_F and H123_EBF1_URT_R primers18 (Supplementary Table 1), which have a 25-bp overlap with the erVenus and attL2 sequence, respectively (EBF1 3′ UTR). erVenus/pDONR plasmid (30 ng) was amplified with the EBF1 3′ UTR PCR product (600 ng) by PCR. The amplified PCR product was digested with DpnI enzyme at 37 °C for 2 h and was used for transformation into Escherichia coli (DH5α) by electroporation (erVenus–EBF1 3′ UTR/pDONR). RPS5A:erVenus–3AT/VD8034GW-mTurq and RPS5A:erVenus–EBF1UTR–3AT/FRm43GW were generated from RPS5A/pDONR, 3AT/pDONR, FRm43GW, VD8034GW-mTurq and erVenus/pDONR or erVenus–EBF1 3′ UTR/pDONR, respectively, by MultiSite Gateway LR clonase reaction.
From the Col-0 genome, promoter sequences of 1.1 kilobases (kb) of PCO1 (from −958 to +198) and 2.5 kb of PCO2 (from −2213 to +298) were amplified by PCR using H68_proPCO1_F and H69_proPCO1_R, or H70_proPCO2_F and H71_proPCO2_R primers, respectively23 (Supplementary Table 1). The amplified PCR products were cloned into the p1R4z-pDONR vector by BP reaction (proPCO1/p1R4z-pDONR, proPCO2/p1R4z-pDONR). proPCO1:erVenus–3AT/FRm43GW and proPCO2:erVenus–3AT/FRm43GW were generated from erVenus/pDONR, 3AT/pDONR41, FRm43GW42, and proPCO1/p1R4z-pDONR or proPCO2/p1R4z-pDONR, respectively, by MultiSite Gateway LR clonase reaction.
The plasmids generated in this work were introduced into Col-0.
Surgical injury of the periderm and chemical treatment
Roots within 5 mm below the root–hypocotyl junction were used for the wounding experiment unless stated otherwise. Under a dissection microscope, the shoot was pulled upwards slightly to create tension in the roots; the roots were longitudinally cut with a razor blade. As roots where the cut reached the vascular cambium region tended to form a callus-like structure instead of the wound periderm at 4 dai, we focused on sections in which the depth of the cut reached between the phloem parenchyma contacting the periderm to the phloem-side cambium in the following analysis unless stated otherwise. For the analysis of promoter induction at the wound site, we also excluded the sections in which the cut was just at the primary phloem pole because reporter expression tended not to be induced. To peel off the periderm for the oxygen level measurement, we made a shallow cut on the surface of the roots tangentially; the cut edge at the wound site was grasped with forceps and pulled towards the root tip.
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