Download nph4277-sup-0001-FigS1-S7

Figure S1. Effects of AVG, DIECA, DPI, NMMA, STA, and OKA on IbRPK expression in
sweet potato (Ipomoea batatas cv. Tainung 57).
Leaves with petiole cuts were immersed in water for 12 h, and then treated water, 0.88 mM AVG
(a), 10 mM DIECA (a), 100 μM DPI (b), 500 μM NMMA (b), 1 μM STA (c), or 0.5 μM OKA (c)
for another 12 h. Then, these leaves were unwounded (W-) or wounded (W+) by tweezers. The
total RNAs of these leaves were analyzed by RT-PCR 0.5 h later. AVG is an ethylene
biosynthesis inhibitor, DIECA is a JA biosynthesis inhibitor, DPI is a NADPH oxidase inhibitor,
NMMA is a NOS inhibitor, STA is a protein kinase inhibitor, and OKA is a protein phosphatase
inhibitor. The expression of IbActin in each assay was also analyzed by RT-PCR, and was treated
as a loading control.
Figure S2. Secondary structure and sequence comparison of miR828 precursor.
(a) Secondary structure of miR828 precursor was predicted by mfold (Zuker, 2003). The line
indicates the position of the mature miR828. (b) Sequence comparison of miR828. Sequence
comparisons were produced by MEGALIGN. Sequences identical are displayed as white letters
in black boxes. Ib-premiR828 was compared with those encoding premiR828 in Arabidopsis
thaliana, Arabidopsis lyrata, Salvia sclarea, and Vitis vinifera.
Figure S3. Putative conserved domains in IbRPK, IbTLD, and IbMYB.
NCBI Conserved Domain Database (CDD) was used. (a) IbRPK contains Leucine rich repeat N-
terminal domain and catalytic domain of protein kinase. (b) IbTLD contains TLDc domain. (c)
IbMYB contains SANT domain.
Figure S4. The expression patterns of IbMYB-siRNA, IbTLD-siRNA, and miR828 in the leaves
of wild type sweet potato (Ipomoea batatas cv. Tainung 57) upon wounding.
The third fully expanded leaves were wounded by tweezers for 0 (W-), 0.5, 1, 3, and 6 h. The
total RNAs from wounded leaves at the time indicated were analyzed by northern blotting for
detecting miR828, IbMYB-siRNA (a), IbTLD-siRNA (b), and 5S rRNA. The values of miR828
and siRNA were adjusted by their corresponding amount of 5S rRNA for equality of loading.
After the adjustment by 5S rRNA, the reaction with the unwounded leaves was treated as the
normalized reference, with a value of one, for determining the relative amount of miR828 and
siRNA.
Figure S5. Phylogenetic analyses of IbMYB and IbTLD.
Phylogenetic trees were produced by MEGA 4.0 programs. (a) Phylogenetic analysis of IbMYB
protein. IbMYB was compared with those encoding other R2R3-type MYB factors in
Arabidopsis thaliana and Ipomoea batatas, which include AtMYB4 (NP_195574), AtMYB7
(NP_179263), AtMYB6 (NP_192684), AtMYB32 (NP_195225), AtMYB1 (NP_187534),
AtMYB113 (NP_176811), AtMYB90 (NP_176813), AtMYB114 (NP_176812), AtMYB75
(NP_176057), and IbMYB1 (BAG68212). (b) Phylogenetic analysis of IbTLD protein. IbTLD
was compared with those encoding similar proteins in various plant species, which include
At4g39870 (NP_195697) from Arabidopsis thaliana, RCOM 0212730 (XP_002531255) from
Ricinus communis, POPTRDRAFT 207435 (XM_002307051) and POPTRDRAFT 832269
(XM_002310528) from Populus trichocarpa, LOC100262287 (XM_002267254) from Vitis
vinifera, and Os02g0754000 (NP_001048151) and Os06g0221100 (NP_001057175) from Oryza
sativa.
Figure S6. Protein sequence comparisons of miR828 targets, IbMYB and IbTLD.
Figure S6. Protein sequence comparisons of miR828 targets, IbMYB and IbTLD. (continued)
Protein sequence comparisons were produced by MEGALIGN. Amino acid residues identical to
those in target proteins are displayed as white letters in black boxes. (a) IbMYB was compared
with those encoding R2R3-type MYB factors in Arabidopsis thaliana and Ipomoea batatas,
which include AtMYB4 (NP_195574), AtMYB7 (NP_179263), AtMYB6 (NP_192684),
AtMYB32 (NP_195225), AtMYB1 (NP_187534), AtMYB113 (NP_176811), AtMYB90
(NP_176813), AtMYB114 (NP_176812), AtMYB75 (NP_176057), and IbMYB1 (BAG68212).
(b) IbTLD was compared with those encoding similar proteins in various plant species, which
include At2g05590 (NP_849938) and At4g39870 (NP_195697) from Arabidopsis thaliana,
RCOM 0212730 (XP_002531255) from Ricinus communis, POPTRDRAFT 207435
(XM_002307051) and POPTRDRAFT 832269 (XM_002310528) from Populus trichocarpa;
LOC100262287 (XM_002267254) from Vitis vinifera; and Os02g0754000 (NP_001048151) and
Os06g0221100 (NP_001057175) from Oryza sativa.
Figure S7. The expression patterns of the phenylpropanoid pathway genes and antioxidant
enzyme genes in transgenic tobacco (Nicotiana tabacum L. cv. W38) overexpressing
IbMYB and IbTLD.
The total RNAs from Wild-Type (W38) and transgenics overexpressing IbMYB (IbMYB-1
and IbMYB-2) or IbTLD (IbTLD-1 and IbTLD-2) were used to detect the phenylpropanoid
pathway genes, antioxidant enzyme genes, and NtActin expression using quantitative RTPCR. The expression levels of NtActin were used as controls for quantitative comparison.