Table S1. List of primers used in this study.

Transcription

1 Gene/Construct/ mutant Primer name Sequence (5 ->3 ) Confimation of T-DNA insertion clh1-1 LBb1 5 GCGTGGACCGCTTGCTGCAACT3 1LP1 5 CCGAAAATGATAAATGCATGG3 1RP1 5 ATGTCCAGCTCGAAAGATTCC3 clh2-1 Lb4 5 CTACAAATTGCCTTTTCTTATCGAC3 PD415 5 ATTTGATTTTGTTTATGTGCTCTT3 PD414 5 GCTGAGAATAGAAGGAGTTGTAGA3 pph2 PPH-for2 5 CAATCATGCTTGCTCCTGGTG LP 5 CTACCAATCCTGGACTCCTCC 3 Spm32 5 TACGAATAAGAGCGTCCATTTTAGAGTGA 3 pph RP 5 TGTACAGGTTATCGGTGAGCC LP 5 CTACCAATCCTGGACTCCTCC 3 Gabi-LB 5 CCCATTTGGACGTGAATGTAGACAC 3 Real-Time PCR VTE5 VTE5-LP 5 GCAGGAAGCATCTCCATGTT3 VTE5-RP 5 GGTCGTTTCCCAGTTCATGT3 PES2 PES2-LP 5 CTGCGAAATTGTCCTGGATT3 PES2-RP 5 TCTCATCGCCTTCCCTTATG3 HPT HPT-LP 5 GTTGTCGCAAAACCGAAGTT3 HPT-RP 5 CAGTGGCATTAACCCGAAAT3 NYE1 NYE-LP 5 TGGGCAAATAGGCTATACCG3 NYE-RP 5 CCACCGCTTATGTGACAATG3 PPH PPH-LP 5 TGAGACCGTTATGGGGAAAG3 PPH-RP 5 CCTCAGGGACTTCATCGTGT3 HPPD HPPD-LP 5 GGCGGCTTGACCACGCCGTG3 HPPD-RP 5 CGGCGGTTCCAACGTCGTCT3 RT-PCR CLH1 CLH1-S 5 CGAAGTGGAACAACGTGATG3 CLH1-AS 5 AGAAACGCAACCACAATTCC3 CLH2 CLH2-S 5 ATGTCCTCTTCTTCATCAAGAA3 CLH3-AS 5 TTGGAAGGTAAGCCAACACC3 PPH2 PPH-for2 5 CAATCATGCTTGCTCCTGGTG LP 5 CTACCAATCCTGGACTCCTCC 3 PPH RP 5 TGTACAGGTTATCGGTGAGCC LP 5 CTACCAATCCTGGACTCCTCC 3 Actin ACT2-S 5 TGGAATCCACGAGACAACCTA3 ACT2-AS 5 TTCTGTGAACGATTCCTGGAC3 Table S1. List of primers used in this study.

2 Figure S1 CLH1 CLH2 PPH2 PPH3 Actin2 Figure S1. RT-PCR analysis of CLH1, CLH2, and PPH gene expression in developmental stage of silique from different mutants. Actin2 was used as control.

3 Figure S WT PPH-4 PPH-3 PPH-2 Figure S2. Expression of PPH in wild type (WT) and PPH-overexpressing lines. s of PPH in seeds of WT and three independent lines seed-specifically over-expressing PPH were normalized to AT5G Data represent mean and SE of at least three replicates.

4 Figure S WT PPH-1 Figure S3. Expression of PPH in wild type (WT) and a PPH-overexpressing line during seed development at 6-,9-,12-,15-days after pollination (DAP). s of PPH were normalized to AT5G Data represent mean and SE of at least three replicates.

5 Seeds Stage 6w/o Siliques Seeds Stage 7w/o Siliques Seeds Stage 8w/o Siliques Seeds Stage 9w/o Siliques Seeds Stage 10w/o Siliques Figure S4 TMT TC (At1g64970) (At4g32770) value PPH (At5g13800) VTE5 (At5g04490) HPPD (At1g06570) GGR (At4g38460) ATHPT (At2g18950) Figure S4. Schematic representation of relative expression of tocopherol synthesis relevant genes from publicly available transcriptomic datasets from Arabidopsis developing seeds. Seed developmental stages 6 to 10 without siliques are shown.

6 A HPPD VTE5 Figure S5 B PES2 WT HPPD WT * PPH-1 PPH-1 PES2 * HPT VTE5 WT PPH-1 HPT Figure S5. Analysis of expression of some genes involved in tocopherol biosynthesis in seeds.the expression of the indicated genes was determined by real-time PCR in seeds of WT and a PPH-overexpressing line (PPH-1) at 6-, 9-, 12- and 15-DAP for HPPD,VTE5,PES2 and HPT. (A) and (B) are two different biological replications. Data represent mean and SE of three technical measurements. Gene expression was normalized to AT5G09810.P<1, *P<5; Students s t-test. 6-DAP 9-DAP 12-DAP 15-DAP 6-DAP 9-DAP 12-DAP 15-DAP

7 Figure S6 A B C D Figure S6. Genotyping of different mutants by PCR. Primer pairs (see Table S1) specifically detecting the T-DNA insertions of clh1-1 (A), clh2-1 (B), pph-2 (C) and pph-3 (D) were used for genotyping of different mutants. The upper lane of each panel shows PCR products amplified with gene-specific primers, while the lower lane shows PCR amplification with the respective right gene-specific primer and the left T-DNA border primer.

8 Chlorophyll content (mmol/g fresh weight) Figure S7 A B Figure S7. Characterization of simultaneous absence of CLHs and PPH during dark-induced senescence.(a) Leaves of wild type (WT) and the different mutants indicated in panel after 5 d of treatment in darkness. (B) Chlorophyll quantification of leaves like shown in panel (A). Data in (A) and (B) represent mean and SE of at least three replicates. P<1, Students s t-test.

9 Figure S WT-1 NYE1-3 NYE1-4 NYE1-2 Figure S8. Expression of NYE1 in wild type (WT) and NYE1-overexpressing lines. s of NYE1 in seeds of WT and three independent lines seed-specifically over-expressing NYE1 were normalized to AT5G09810.