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Supporting Information
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Figure S1. The 252-bp DNA fragment upstream from the start codon of PAO is a
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functional promoter.
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(a) GUS activities of different transgenic lines driven by a series of 5’-truncated PAO
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promoter fragments. Left panel: 1200-, 550-, 252- and 100-bp fragments upstream
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from the start codon of PAO fused to the GUS gene. Col-0 and p35S:GUS transgenic
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plants were controls. Right panel: GUS activities in plants with or without 48 hr of
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dark treatment. Fold induction after dark treatment is on the right of columns.
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(b) RT-qPCR of mRNA level of PAO in homozygous transgenic plants. RNA was
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extracted from 5th and 6th detached leaves of each line. β-ACTIN2 was an internal
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control. Data are means±SD of 2 independent experiments. *** P <0.001 comparing
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transgenic plants and acd1-20 (t-tests).
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(c) Chl contents in the detached leaves of different p252:PAO acd1-20 transgenic lines
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as well as Col-0 and acd1-20 plants. Detached leaves of each genotype were treated in
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darkness for five days, and their Chl contents were measured afterwards.
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(d) HPLC examination of Chl catabolites in the leaves of p252:PAO acd1-20 #1 as
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well as Col-0 and acd1-20 plants. Detached leaves from plants of each genotype were
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dark-treated for 5 days. Pheide a, pheophorbide a; Chl a, chlorophyll a; Chl b,
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chlorophyll b.
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In (a) and (c), data are mean±SD of 3 biological replicates. * P <0.05, *** P <0.001
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(t-tests).
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Figure S2. The PAO promoter is responsive to MeJA treatment.
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(a) p252:PAO acd1-20 #1 transgenic plants restored the stay-green phenotype of
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acd1-20. The 5th and 6th detached leaves from 4-week-old Col-0, acd1-20 and
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p252:PAO acd1-20 #1 transgenic plants were treated with or without 100 µM MeJA
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for 4 days.
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(b) Chl contents in detached leaves shown in (a).
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(c) RT-qPCR of mRNA level of PAO in detached leaves shown in (a). β-ACTIN2 was
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an internal control.
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Data are mean±SD of 3 biological replicates. *** P <0.001 (t-tests).
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Figure S3. Attached leaves of Col-0 and myc2myc3myc4 show a phenotype
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concordant to those of their detached counterparts after MeJA treatment.
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(a) Phenotypes of myc2myc3myc4 (myc2, 3, 4) as well as Col-0 plants after MeJA
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treatment. 4-week-old plants of Col-0 and myc2, 3, 4 were treated with or without
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1mM MeJA for 6 days before photographing.
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(b) Chl contents in 5th and 6th rosette leaves of the plants shown in (a). Data are
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mean±SD of 3 biological replicates. *** P <0.001 (t-tests).
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Figure S4. Phenotypes of myc double mutants after MeJA treatment.
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(a) Phenotypes of Col-0 and myc double mutant plants after MeJA treatment.
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4-week-old plants of Col-0 and myc2myc3, myc2myc4, myc3myc4 mutants were
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treated with or without 100 µM MeJA for 4 days. Images showed the 5th and 6th
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detached leaves.
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(b) Chl contents in the 5th and 6th rosette leaves shown in (a). Data are mean±SD of 3
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biological replicates. *** P <0.001 (t-tests).
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Figure S5. Overexpression of MYC2/3/4 accelerates Chl degradation upon MeJA
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treatment.
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(a) Acceleration of leaf yellowing by overexpression of MYC2, MYC3 or MYC4 after
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MeJA treatment. Detached leaves of 35S:MYC2, 35S:MYC3 and 35S:MYC4 as well as
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Col-0 and 35S:GFP were treated with or without 100 µM MeJA for 60 hr before
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photographing.
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(b) Chl contents in the detached leaves shown in (a). Different letters within each
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treatment indicate significant differences at P <0.001 (one-way ANOVA).
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(c) qRT-PCR analysis of mRNA level of PAO in the detached leaves shown in (a).
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β-ACTIN2 was an internal control.** P <0.01, *** P <0.001(t-tests).
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Data are mean±SD of 3 biological replicates.
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Figure S6. Transcript levels of PAO in the inducible transgenic lines in WT and the
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myc2myc3myc4 triple mutant backgrounds.
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RT-qPCR analysis of mRNA level of PAO in each transgenic line in backgrounds of
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Col-0 (a) and myc2myc3myc4 triple mutant (b). β-ACTIN2 was an internal control.
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Data are mean±SD of 2 biological replicates. *** P <0.001 comparing WT and the
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myc2myc3myc4 mutant (t-tests).
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Figure S7. nolnyc1 mutant conferred a stay-green phenotype.
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(a) Phenotypes of the detached leaves of nolnyc1, nol, nyc1 and Col-0 plants after
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MeJA treatment. The 5th and 6th detached leaves of 4-week-old plants were treated
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with or without 100 µM MeJA for 4 days.
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(b) Chl contents in the detached leaves shown in (a). Data are mean±SD of 3
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independent experiments. *** P <0.001 (t-tests).
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Figure S8. ANAC019/055/072 are the targets of MYC2
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(a) RT-PCR analysis of mRNA level of ANAC019/055/072 in myc2myc3myc4 (myc2,
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3, 4) after MeJA treatment. Experimental details were as in Figure 2a. Data are
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mean±SD of 3 biological replicates.
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(b) Association of MYC2-GFP fusion protein with ANAC019 promoter in planta.
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ChIP product was as described in Figure 1c. Distinct regions examined in the
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ChIP-qPCR assay are indicated under the respective column charts."-", upstream of
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ATG. Data are mean±SD of 2 independent experiments. *** P <0.001 (t-tests).
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Figure S9. In vitro pull-down assays of MYC2 and ANAC019.
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About 50µg purified MBP and MBP-MYC2 proteins were mixed with the same
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amount of purified GST-ANAC019 proteins from E.coli and used as input for the
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pull-down assay. GST agarose beads and amylose beads were used to pull down
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MYC2 (a) and ANAC019 (b). The input and pull-down samples were then detected
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with MBP antibody (a) or GST antibody (b).The purified GST-ANAC019 and MBP
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and MBP-MYC2 proteins were stained with Coomassie blue.
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Figure S10. MYC2 and ANAC019 respectively regulate the expression of PAO and
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NYE2.
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MYC2 (a) and ANAC019 (b) regulate the expression of PAO and NYE2 in transient
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dual luciferase assays in Arabidopsis protoplasts. Col-0 protoplasts were
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co-transformed with the pPAO:LUC reporter (a) or pNYE2:LUC reporter (b) construct
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and 35S:MYC2 and/or 35S:ANAC019 effector constructs. Vector, empty vector
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control. Data are mean±SD from 2 biological replicates. *** P <0.001(t-tests).
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S1Table. Primers used in this study.
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