Fig. S1. Amino acid sequence alignment of MYBS3 proteins. MYBS3 protein sequences of Arabidopsisthaliana (MYBH; NP_199550); (At3g16350; NP_188256), Glycine max (GmMYB93; ABH02845), Medicago truncatula (MtMYB48; XP_003608683), Vitis vinifera (VvMYB1R1; XP_002274350), Solanum tuberosum (MYBSt1; AAB32591), Sorghum bicolor (Sb01g029020; XP_002464930), Zea mays (ZmMYBSt1; NP_001266509), Oryza sativa (OsMYBS3; AAN63154); (Os01g018790; NP_001042254) and Hordeum vulgare (HvMYBS3; CAJ53899) were aligned by ClustalW2. Four conserved regions including CCHC zinc finger with R/KLFGV repression domain, MYB with NLS region, NES containing region and EAR motif are indicated by lines above the sequences. NES is indicated by asterisk (*).

Supplementary Fig. S1.

Supplementary Fig. S2.

Fig. S2. Expression pattern of MYBH was induced by darkness and aging. A. RT-PCR and qRT-PCR analysis of MYBH expression under darkness treatment for various time periods, total RNA was isolated from 2-week-old treatment with darkness and then subjected to RT-PCR and qRT-PCR analysis. B. Two-week-old MYBHp::GUS-1 and MYBHp::GUS-2 transgenic lines were grown on ½ MS without sucrose medium under light or darkness conditions for 2 days, and then subjected to GUS activity by histochemical staining. C. Gene expression pattern were obtained from the publicly available microarray data at the GENEVESTIGATOR database (https://www.genevestigator.ethz.ch). D. GUS activity analysis in MYBHp::GUS-1 Arabidopsis grown in soil for 14 days, 28 days and 35 days. Bar= 1 cm.

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Supplementary Fig. S3.

Fig. S3. Identification of mybh mutant lines, MYBH overexpression and MYBH-complementation transgenic lines. A, Schematic diagram represents structure of MYBH gene and the construct used for overexpression of MYBH. Block boxes and gray boxes represent coding sequences and untranslated sequences, respectively. The two T-DNA insertion alleles, mybh-1 and mybh-2, are indicated by open triangles. The positions of the primer sequences used for PCR genotyping of MYBH and the mybh mutant alleles are marked with arrows. The primers 3-1 and 3-2 were used to screen the mybh-1 mutants. The primers 3-3 and 3-4 were designed specifically for screening of mybh-2 mutant. The primers S3-5 combined with 3-2 were used to detect the full-length of MYBH mRNA. B and C, RT-PCR analysis for MYBH mRNA in the wild type (WT), the mutants (mybh-1 and mybh-2), the overexpression (MYBHOX-1 and MYBHOX-2) lines and the MYBH complementation (MYBg-Com-1, -2 and -3)lines. RNAs were isolated from 2-weeks-old seedlings and subjected to RT-PCR analysis. Arabidopsis Act1 was used as an internal control.

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Supplementary Fig. S4.

Fig. S4. The bolting time of mybh-1 and MYBH overexpression transgenic lines. WT, mybh-1, and MYBH overexpression lines (MYBHOX-1 and MYBHOX-2) transgenic of Arabidopsis were grown in pots under long day (LD) or short day (SD) for 28 d and 42 d, respectively (A). The bolting time and number of rosette leaves of WT, mybh-1, MYBHOX-1, MYBHOX-2 growing under LD and SD. Error bars indicate the SD; n = 6. * is significantly different from the wild-type plants (Student’s t test: p <0.05).

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Supplementary Fig. S5.

Fig. S5. Leaf senescence of was accelerated in MYBH overexpression lines and delayed in mybh-1 knockout line. WT, mybh-1, and MYBH overexpression lines (MYBHOX-1 and MYBHOX-2) transgenic of Arabidopsis were grown in pots under normal conditions for 35 days. Bar = 5 cm.

c c L1 L2 L8L7L6L5L4L3 L12L11L10L9L13L14

Supplementary Fig. S6.

Fig. S6. Dark-induced detached leaf senescence was accelerated in MYBH overexpression lines and delayed in mybh-1 knockout lines. A, The fourth leaves were detached from 20-day-old soil-grown plants, and then were incubated in the MES medium (pH= 5.8) under darkness for 1 or 5 d. B and C, Chlorophyll contents (B) and quantum yields (C) of dark-treated leaves were analyzed. Error bars indicate the SD; n = 8. * is significantly different from the wild-type plants (Student’s t test: p <0.05).

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Supplementary Fig. S7.

Fig. S7. Expression profiles of up-regulated genes in MYBHOX-1 plants. Genes that expression level were 2-fold more in dark treatment of MYBHOX-1 than WT were analyzed by Gene Ontology (GO) annotation in AGRIgo web-based program (http://bioinfo.cau.edu.cn/agriGO/). P-values were calculated by Fisher’s test to compare the percentage distribution of GO annotation from genes repressed in MYBHOX-1 and the whole genome.

Supplementary Fig. S8.

Fig. S8. Expression profiles of down-regulated genes in MYBHOX-1 plants. Genes that expression level were 2-fold less in dark treatment of MYBHOX-1 than WT were analyzed by Gene Ontology (GO) annotation in AGRIgo web-based program (http://bioinfo.cau.edu.cn/agriGO/). P-values were calculated by Fisher’s test to compare the percentage distribution of GO annotation from genes repressed in MYBHOX-1 and the whole genome.

Dark treatment (hr)

0 12 24 36 48

Supplementary Fig. S9.

Fig. S9. Expression pattern of DFL1 and DFL2 was reduced by darkness. qRT-PCR analysis of DFL1 and DFL2 expression under darkness treatment for various time periods, total RNA was isolated from two-week-old seedling treatment with darkness and then subjected to qRT-PCR analysis. The DIN3 (dark-inducible 3; AT3G06850) was used as a positive control. Gene expression was related to without darkness treated plants, as 1. * is significantly different from the without darkness treated plants (Student’s t test: p <0.05).

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Supplementary Fig. S10.

WT

Fig. S10. Transgenic Arabidopsis overexpressing MYBH–GFP and GFP–MYBH exhibited acceleration of both developmentally regulated and dark-induced leaf senescence. A. Early senescence phenotype shown in MYBH–GFP and GFP–MYBH transgenic lines. The WT, MYBH–GFP #1, GFP–MYBH#1 and GFP–MYBH#2 transgenic lines were grown in pot under normal condition for 35 days. B and C. Acceleration of dark-induced leaf senescence in MYBH–GFP and GFP–MYBH. The fourth leaves of 20-day-old plants of WT, mybh-1, GFP–MYBH #1, #2 and MYBH–GFP #1 were excised and incubated with the MES medium (pH= 5.8) in dark for 3 days. The leaf morphology (B) was observed and the relative chlorophyll content (C) was be compared. Error bars indicate the SD; n = 6. * is significantly different from the wild-type plants (Student’s t test: p <0.05).

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Supplementary Fig. S11.

Fig. S11. Differences in the phenotypes of hypocotyls and roots in wild type, mybh-1, MYBHOX-1 and MYBHOX-2 seedlings. The WT, mybh-1, MYBHOX-1 and MYBHOX-2 were germinated on 1/2 MS medium, and then the seedlings were incubated in 16h light/ 8h dark growth chamber for 12 days. A. A photo of seedlings. B. Quantification of hypocotyl length and number of lateral roots in these seedlings. Error bars indicate the SD; n=20.

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