1. Col
max1-1
max3-9
max4-11
Atd14-1
max2-4
ein2-5
ein3-1 eil1-3
0 day
7days A B * * * * * * * *
Col C
Supplemental Figure S1. Quantitative analysis of senescence parameters in strigolactone biosynthesis mutants. A, Phenotype of strigolactone biosynthesis mutants incubated in the light. The 8th leaf from the top was incubated in the light for 7 days. The lower panel shows changes in SPAD values over time. B, Changes in Fv/Fm values obtained from the 8th leaf incubated in the dark. Fv/Fm values of 7 days after dark treatment were compared with those of Col by using Student’s t-test (*, p < 0.05; **, p < 0.01). C, Changes in membrane ion leakage over time during dark incubation, for which the 11th leaf was used. A–C; Bars indicate standard error (n = 6).

2. A 5’ 3’
kai2-3 B * *
Ler
kai2-3
kai2-3 C
Ler
kai2-3
8th
9th
10th
11th
12th D
Ler
kai2-3
Supplemental Figure S2. Phenotype of kai2 in dark-induced senescence. A, Structure of the kai2-3
allele. Open boxes and horizontal bars indicate exons and introns, respectively. The triangle shows
the Ds transposon insertion. B, Hypocotyl length of Ler and kai2-3 (Ler background) germinated
under dim red light for 4 days. Hypocotyl length of kai2-3 is longer than that of Ler at 1% level by
Student’s t-test, suggesting that kai2-3 is a loss-of-function mutant. Bars indicate standard error (n
= 15). C, Leaves of Ler and kai2-3 were incubated in the dark for 6 days. D, Changes in SPAD values
over time of the 8th leaf during 7 days of dark incubation. Bars indicate standard error (n = 7).
Student’s t-test indicated that no statistical difference was observed between Ler and kai2-3 leaves
in the same day during dark incubation.

3. A B C * * * * * Relative mRNA levels *
Col
Col
max4-11
max2-4
ein3-1 eil1-3 C * *
Relative mRNA levels
MAX3
MAX4
MAX1
AtSGR1 * * * *
Presenescent
Senescing
Senescing
Fully senescent
Presenescent
Fully senescent
Supplemental Figure S3. Natural senescence of strigolactone biosynthesis mutants. A, Delayed-senescence phenotype of max4-11 in 10-week-old (28 days after bolting) plants grown under long-day conditions. B, SPAD values of leaves at 28 days after bolting, where the 11th leaf at bolting was used for measurement. Bars indicate standard error (n = 5). C, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in naturally senescent leaves. Pre-senescent: entirely green; senescing: yellow leaf tip; fully senescent: entirely yellow leaf. Leaves were collected from 10-week-old Col plants (grown under short-day conditions for 4 weeks and long-day conditions for 6 weeks). Transcript levels are relative to that in fully senescent Col leaves. ACT8 was used as a reference. Bars indicate standard error (n = 4). mRNA levels were statistically compared with that of pre-senescent leaves by using Student’s t-test (*, p < 0.05).

4. A
Ethylene
Air a a ab ab ab b c d
Col B
Ethylene
Air
Ethylene
Air
Supplemental Figure S4. Ethylene sensitivity of strigolactone biosynthesis/strigolactone-insensitive and ethylene-insensitive mutants in leaf senescence. A, Chlorophyll content of strigolactone biosynthesis and strigolactone-insensitive mutants treated with or without ethylene in the light for 7 days. Solid and open bars indicate air- and ethylene-treated leaves, respectively. SPAD values of ethylene-treated leaves were statistically compared by using Tukey’s multiple comparison method. Data indicated with the same letter are not significantly different (p < 0.05). B, Changes in chlorophyll content over time in ein2-5 and ein3-1 eil1-3 during 20 days of ethylene treatment in the light. No statistical difference was observed between air- and ethylene-treated leaves in the same day after dark treatment by using Student’s t-test. The 8th leaf was used. Bars indicate standard error. n = 6 in A and n = 8 in B.

5. A B MAX4 AtSGR1 Relative mRNA levels MAX3 AtSGR1 Relative mRNA levels
Col
max3-9
Col
max3-9 B
MAX3
AtSGR1
Relative mRNA levels
Col
max4-11
Col
max4-11
Supplemental Figure S5. MAX3 and MAX4 expression in max4-11 and max3-9 during dark incubation. qRT-PCR analysis of transcript levels of MAX4 in max3-9 (A) and MAX3 in max4-11 (B). The 8th leaf of Col, max3-9, and max4-11 was incubated in the dark for 7 days. Transcript levels are relative to that in Col leaves at 7 DDT. ACT8 was used as a reference. Bars show standard error (n = 4).

6. Membrane ion leakage (%)C a a a a a a
Chlorophyll content (SPAD) b a a a c a b b a a a a a a
Fv/Fm b a a b a a a
Membrane ion leakage (%) a b a b a a a b b
Supplemental Figure S6. Senescence parameters in ein3-1 eil1-3 leaves during a long dark incubation. A, Changes in SPAD values over time during dark incubation. B, Changes in Fv/Fm values over time during dark incubation. C, Changes in membrane ion leakage over time during dark incubation. A–C; Bars indicate standard error. n = 8 in A and C. n = 6 in B. Data indicated with the same letter are not significantly different by Tukey’s multiple comparison method (p < 0.05).

7. Days after dark treatment
MAX3
MAX4
Relative mRNA levels
MAX1
AtSGR1
Col
ein3-1 eil1-3
Col
ein3-1 eil1-3
Days after dark treatment
Supplemental Figure S7. qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in senescent ein3-1 eil1-3 leaves. Transcript levels are relative to that in Col leaves at 7 DDT. ACT8 was used as a reference. Bars indicate standard error (n = 4).

8. A B C ein3-1 eil1-3 Atd14-1 * * * * * * * * * * * Col ERF1 EBF2
Mock
GR24 A
8th
9th
10th
11th
12th B
Mock
GR24 *
Chlorophyll content (SPAD） * * * * * *
Col
ein3-1 eil1-3
Atd14-1 C * * *
ERF1 *
EBF2
Relative mRNA levels
Col
ein3-1 eil1-3
Col
ein3-1 eil1-3
Supplemental Figure S8. Effect of strigolactone on senescence in lower leaves. A, Strigolactone promotes senescence of lower leaves in Col, but not in ein3 eil1, in the light. Lower (8–12th) leaves were incubated with or without 25 µM GR24 in the light for 7 days. B, Chlorophyll content in the lower leaves of Col and ein3-1 eil1-3 treated with or without 25 μM GR24 in the light. Solid and open bars indicate mock and GR24-treated leaves, respectively. Chlorophyll contents of mock and GR24-treated leaves of the same leaf position were statistically compared by using Student’s t-test. C, qRT-PCR analysis of transcript levels of ethylene-inducible genes in lower leaves of Col grown under the short-day condition. ERF1 and EBF2 are direct targets of EIN3. Student’s t-test revealed that ERF1 and EBF2 were significantly upregulated in the 12th leaf of Col but not in ein3-1 eil1-3. Transcript levels are relative to that in the 12th leaf of Col. ACT8 was used as a reference. Bars indicate standard error. n = 6 in B and n = 4 in C. *, p < 0.05; **, p < 0.01.

9. A
max4-11
Col
max4-11 7
Genomic DNA M B
MAX4
ACT8
Supplemental Figure S9. Characterization of the max4-11 allele. A, Structure of the max4-11 allele. Open boxes and horizontal bars indicate exons and introns, respectively. The triangle shows T-DNA. B, Expression of MAX4 in the max4-11 mutant. Upper panel shows RT-PCR analysis of MAX4 using MAX4F (F) and MAX4R (R) as a primer pair. Lower panel shows expression of ACT8 as a reference. “0” and “7” indicate 0 and 7 DDT.

10. Supplemental Figure S10. Effect of strigolactone on Fv/Fm value
Supplemental Figure S10. Effect of strigolactone on Fv/Fm value. The 8th leaf from the top of Col plants were incubated in the light for 7 days with 25 μM GR24. No statistical difference was observed between mock and GR24-treated leaves by using Student’s t-test. n = 7

11. Supplemental Table 1. Primers for genotyping
Sequence
max2-4 F
　CGAGGCTTACACGGTTAGAT
max2-4 R
　CACAAATCGGCTCAATTGCC
max4-11 F
　GTAACTTCCCCAATGCTCTC
max4-11 R
　CAGAGAGGAGAAGTGTCTGA
At d14-1 F
　ACACCTCTCAGATCGCTGTT
At d14-1 R
　CAACGCATGCTCTTGTATCC
eil1-3 F
　TAAGTGTAAAGAAGGCGTCGA
eil1-3 R
　ATGACTCGGGATAAAGCTCC
Lba1_SALK
　TGGTTCACGTAGTGGGCCATCG
DsLox_T-DNA
　CCATGTAGATTTCCCGGACA
max3-9 F
　TTAACGGTCACTTGCAACGC
max3-9 R
　GAATTCCGAATCATACTCTGTA
kai2-3 F
　CCAACTATTCGAAGCCATCC
kai2-3 R
　AGTATAGCACAAGCACTCTTG

12. Supplemental Table 2. Primers for dCAPS analysis
Sequence
Restriction Enzyme
max1-1 F
　GTGATGAACACAACAAGTGTC
BlnI
max1-1 R
　CCTTTCTTGTGAAGAGGAGAAGCTGAGCCT
ein2-5 F
　TGTTCTTGATTGTTGATCACAG
RsaI
ein2-5 R
　GAAGCCAAGCGGGTATTTCTATCTTCAGTA
ein3-1 F
　TTGGACCGACTCCTCATACC
AluI
ein3-1 R
　TTGAGGCCACCAATCCTCTTTCCCATTAGC
ctr1-1 F
　CAAAAAGGCGTTAATCTGCTG
EcoRI
ctr1-1 R
　CGTGCTGGCCTTCAATCGCGAGAGACCGAA

13. Supplemental Table 3. Primers for qRT-PCR or RT-PCR
Sequence
Efficiency
ACT8 F
　AGCACTTTCCAGCAGATGTG
1.00
ACT8 R
　GAAATGTGATCCCGTCATGG
MAX1 F
　GGCCCTATTTTCAGATTTCAG
0.96
MAX1 R
　GAAGGAATGCTTCTGTTTGGA
MAX3 F
　AAAGACTTTTAACGGTCACTTG
0.99
MAX3 R
　TTGAATTCCGAATCATACTCAC
MAX4 F
　GATGCTAGGATCGGGAGATT
0.97
MAX4 R
　TGCACATATCCATCGCTCTC
AtSGR1 F
　GATTGTTCCCGTTGCAAGGT
AtSGR1 R
　AAGTCCTAGGGAGCGTTGAA
SAG12 F
　CTATTACAGGTTATGAGGATGT
SAG12 R
　AAACCACCTCCTTCAATTCCA
ERF1 F
　CGATCCCTAACCGAAAACAG
0.98
ERF1 R
　TCCGATAGAATATTCCGGTGA
EBF2 F
　TCAGATTTAGTGGTGATGAAGA
EBF2 R
　GTAATATACACCGGGACAGC
RT_ACT8 F
　CAGCATGAAGATTAAGGTCGT
　　　-
RT_max4-11 F
　GCACGAGCATGGTATGATAC
RT_max4-11 R
　CAACCATGCAAGCCATAAGG