2024年3月30日发(作者：公羊良骏)

Supplementary Material

Fig S1. Interactions of OsDof24 with the OsC4PPDK promoter in rice protoplasts

(a) Loss-of-function analysis of the OsC4PPDK promoter Effects of OsDof24

overexpression and the mapping of OsDof24-binding fragments on the OsC4PPDK promoter

were tested using an overexpression construct Pro35S::OsDof24 which was co-transformed

into rice protoplasts with a series of OsC4PPDK promoter deletion GUS constructs. GUS

activities of co-transformation with Pro35S::OsDof24 are indicated in black and columns

representing empty effector plasmids are blank.

(b) Gain-of-function analysis of OsDof25 with wild type and mutant fragments (-385 to

-274) from the OsC4PPDK promoter The GUS reporter construct ProPPDK-A::GUS bears

the wild type fragment (-384 to -274) from the OsC4PPDK promoter containing motif CTTT.

In constructs ProPPDK-B::GUS, ProPPDK-C::GUS, ProPPDK-D::GUS and ProPPDK-

E::GUS the wild type motif CTTT is mutated into GTTT, CATT, CTAT and CTTA,

respectively. Plasmid pGusXX-47 was used as a negative control for the reporter. In both

panels, the reporter plasmids were co-transformed with Pro35S::OsDof24, or empty effector

pRT100. Relative GUS activities were normalised for total protein. The bar graphs are based

on the mean values of three independent transformations of each construct combination and

error bars represent the standard deviation (SD) of biological replicates. The data were

analysed using ANOVA followed by Bonferroni corrections. Asterisks indicate significant

differences (p<0.05) compared with the untransformed controls.

Page 1 of 8

Supplementary Material

Fig S2. Molecular analysis of OsDof25 RNAi plants

(a) Southern blotting results showing the T-DNA insertion copy number. The hpt gene was

used as probe. (b) Analysis of OsDof25 expression in T

1

OsDof25 RNAi plants (lines #5 and

#30) and control plants, azygous plants separated from the T

0

(WT). (c) Analysis of

OsC4PPDK expression in T

1

OsDof25 RNAi plants (lines #5 and #30) and control plants,

which are azygous plants separated from the T

0

(WT). Ubiquitin and Actin genes were used

for equilibration of cDNA quantity in qPCR experiments. Bars represent means standard

error (n=3 independent qPCRs). The bargraphs are based on the mean values of three

independent transformations of each construct combination and error bars represent the

standard deviation (SE) of biological replicates. The data were analysed using ANOVA

followed by Bonferroni corrections. Asterisks indicate significant differences (p<0.05)

compared with the untransformed controls.

Page 2 of 8

2024年3月30日发(作者：公羊良骏)

Supplementary Material

Fig S1. Interactions of OsDof24 with the OsC4PPDK promoter in rice protoplasts

(a) Loss-of-function analysis of the OsC4PPDK promoter Effects of OsDof24

overexpression and the mapping of OsDof24-binding fragments on the OsC4PPDK promoter

were tested using an overexpression construct Pro35S::OsDof24 which was co-transformed

into rice protoplasts with a series of OsC4PPDK promoter deletion GUS constructs. GUS

activities of co-transformation with Pro35S::OsDof24 are indicated in black and columns

representing empty effector plasmids are blank.

(b) Gain-of-function analysis of OsDof25 with wild type and mutant fragments (-385 to

-274) from the OsC4PPDK promoter The GUS reporter construct ProPPDK-A::GUS bears

the wild type fragment (-384 to -274) from the OsC4PPDK promoter containing motif CTTT.

In constructs ProPPDK-B::GUS, ProPPDK-C::GUS, ProPPDK-D::GUS and ProPPDK-

E::GUS the wild type motif CTTT is mutated into GTTT, CATT, CTAT and CTTA,

respectively. Plasmid pGusXX-47 was used as a negative control for the reporter. In both

panels, the reporter plasmids were co-transformed with Pro35S::OsDof24, or empty effector

pRT100. Relative GUS activities were normalised for total protein. The bar graphs are based

on the mean values of three independent transformations of each construct combination and

error bars represent the standard deviation (SD) of biological replicates. The data were

analysed using ANOVA followed by Bonferroni corrections. Asterisks indicate significant

differences (p<0.05) compared with the untransformed controls.

Page 1 of 8

Supplementary Material

Fig S2. Molecular analysis of OsDof25 RNAi plants

(a) Southern blotting results showing the T-DNA insertion copy number. The hpt gene was

used as probe. (b) Analysis of OsDof25 expression in T

1

OsDof25 RNAi plants (lines #5 and

#30) and control plants, azygous plants separated from the T

0

(WT). (c) Analysis of

OsC4PPDK expression in T

1

OsDof25 RNAi plants (lines #5 and #30) and control plants,

which are azygous plants separated from the T

0

(WT). Ubiquitin and Actin genes were used

for equilibration of cDNA quantity in qPCR experiments. Bars represent means standard

error (n=3 independent qPCRs). The bargraphs are based on the mean values of three

independent transformations of each construct combination and error bars represent the

standard deviation (SE) of biological replicates. The data were analysed using ANOVA

followed by Bonferroni corrections. Asterisks indicate significant differences (p<0.05)

compared with the untransformed controls.

Page 2 of 8