Figure S1. Expression of Cold-Responsive Genes in myb96-ox and myb96-1 Mutants.
Ten-day-old seedlings grown at 23oC under LDs were harvested for total RNA isolation.
Transcript accumulation was analyzed by RT-qPCR. Biological triplicates were averaged.
Bars indicate standard error of the mean.
Figure S2. Effects of Long-Term Cold on CBF Expression in myb96-1. Ten-day-old
seedlings grown at 23oC under LDs were exposed to cold for the indicated time period (d).
Transcript accumulation was analyzed by RT-qPCR. Biological triplicates were averaged.
Bars indicate standard error of the mean.
Figure S3. Cold Induction of CBF and COR Genes in myb96-ox. Ten-day-old seedlings
grown at 23oC under LDs were exposed to cold for the indicated time period (h). Transcript
accumulation was analyzed by RT-qPCR. Biological triplicates were averaged. Statistically
significant differences between the wild-type and myb96-ox at corresponding time points are
indicated by asterisks (Student’s t-test, *P<0.05). Bars indicate standard error of the mean. M,
mock; C, cold.
Figure S4. Effects of ABA and Cold on Expression of Five HHP Genes. Ten-day-old
seedlings grown at 23oC under LDs were either exposed to cold (C) or transferred to MS
liquid medium supplemented with or without 20 MABA (A) for the indicated time period
(h). Transcript accumulation was analyzed by RT-qPCR. Biological triplicates were averaged.
Bars indicate standard error of the mean. M, mock.
Figure S5. Effects of ABA on HHP Expression in myb96-1. Ten-day-old seedlings grown
at 23oC under LDs were transferred to half-strength MS-liquid medium supplemented with or
without 20 MABA for the indicated time period (h). Transcript accumulation was analyzed
by RT-qPCR. Biological triplicates were averaged. Bars indicate standard error of the mean.
M, mock.
Figure S6. Chromatin Immunoprecipitation (ChIP) Assays using Antibody-Free Resin.
The putative MYB binding sites are indicated by arrowhead. Black lines indicate the regions
for PCR amplification. The ChIP analysis was performed with resin alone. Association of
MYB96 to putative MYB-binding regions of HHP1 (a), HHP2 (b), and HHP3 (c) promoters
was analyzed by qPCR. Biological triplicates were averaged. Bars indicate standard error of
the mean.
Figure S7. Lack of HHP Genes in Corresponding T-DNA Insertional Mutants. (a) to (c)
Mapping of the T-DNA insertion site in the hhp1-1 (a), hhp2-1 (b), and hhp3-1 (c) mutants.
White arrows indicate exons of the corresponding genes. Black arrowheads indicate T-DNA
insertion site. (d) to (f) Transcript accumulation of HHP genes in corresponding T-DNA
mutants. Ten-day-old seedlings grown at 23oC under LDs were harvested for total RNA
isolation. Transcript accumulation was analyzed by semi-quantitative RT-PCR. The
TUBULIN BETA CHAIN 2 (TUB) gene (At5g62690) was used as an internal control.
Figure S8. Effects of ABA on Expression of CBF Genes in hhp Mutants. Ten-day-old
seedlings grown at 23oC under LDs were transferred to half-strength MS-liquid medium
supplemented with or without 20 MABA for the indicated time period (h). Transcript
accumulation was analyzed by RT-qPCR. Biological triplicates were averaged. Statistically
significant differences between the wild-type and mutants at corresponding time points are
indicated by asterisks (Student’s ttest, *P<0.05). Bars indicate standard error of the mean. M,
mock.
Table S1. Primers Used in RT-qPCR and Subcloning. RT-qPCR primers were designed using
the Primer Express Software installed into the Applied Biosystems 7500 Real-Time PCR
System. The sizes of PCR products ranged from 80 to 300 nucleotides in length. F, forward
primer; R, reverse primer.
Table S2. Primers Used in ChIP Assays. F, forward primer; R, reverse primer.