Supporting Information
Figure S1. Expression pattern of miR408 in Arabidopsis development. (a) qRT-PCR analysis
of the relative expression levels of miR408 by three independent experiments. Each
experiment contains three technical replicas. 5S rRNA was used as the endogenous control.
Error bars represent SD (n=3). Experiment 1 is shown in the main text. (b) RNA blot analysis
of mature miR408 in development by three independent experiments. U6 snRNA was used as
a loading control. Experiment 1 is shown in the main text. (c) Transgenic plants expressing
either the pMIR408:GUS reporter gene or the vector were grown on MS plate under
continuous light for seven days and stained for GUS activity. (d) GUS staining in stems of
seven-week-old plants transformed with either the vector or the pMIR408:GUS reporter gene.
The same stem was separated into the upper part and lower part for comparison.
Figure S2. Characterization of additional transgenic plants over-expressing miR408. (a) RNA
blot analysis of the over-accumulation of miR408 in two individual miR408-OX lines in
addition to the line shown in Figure 2 of the main text. U6 snRNA was used as a loading
control. (b) qRT-PCR analysis of miR408 target genes Plantacyanin and LAC13 in wild type
and miR408-OX transgenic seedlings. Actin7 was used as endogenous control. Error bars
present SD (n = 3). (c) Enlarged morphology of miR408-OX transgenic lines compared to
wild type. Shown are 7-day-old seedlings grown on MS under continuous light and 3-weekold adult plants grown in soil under long day condition (L:D = 16:8). Bar = 1 cm. (d)
Quantitative analysis of fresh weight, root length, leaf length and leaf width in wild type and
miR408-OX transgenic plants. Fresh weight and root length were measured with 7-day-old
seedlings grown on MS under continuous light. Leaf length and width were measure with the
fully expanded fifth rosette leaf of adult plants grown in soil under long day condition (L:D =
16:8). Error bars present SD (n ≥ 20). * p < 0.01 and ** p < 0.001 vs wild type by Student’s t
test.
Figure S3. Analysis of T-DNA insertion lines related to the MIR408 and At2g47020 loci. (a)
Genomic location of the seven insertion lines SALK_023586, SALK_038860,
SALK_081087, SALK_082709, FLAG_481E04, and FLAG_545E03 relative to the
overlapping MIR408 and At2g47020 loci. Note that MIR408 and At2g47020 (Peptide chain
release factor 1) are on complementary strands. Positions of the primers used to monitor
At2g47020 transcript levels are also indicated. (b) qRT-PCR analysis of At2g47020
expression level in the SALK and FLAG T-DNA insertion lines using two different primer
pairs as indicated. Actin7 was used as endogenous control. Error bars present SD (n = 3). (c)
qRT-PCR analysis of mature miR408 level in wild type and the seven T-DNA insertion lines.
5s rRNA was used as endogenous control. Error bars present SD (n = 3). (d, e) Morphological
comparison of wild type and the seven T-DNA insertion lines at the seedling (d) and adult (e)
stages. Bar = 1 cm. (f) Quantitative analysis of fresh weight, root length, leaf length and
width in wild type and individual T-DNA lines. Leaf measurements were performed on the
fully expanded fifth rosette leaf of adult plants grown in soil under long day condition (L:D =
16:8). Error bars present SD (n ≥ 20). * p < 0.01 and ** p < 0.001 vs wild type by Student’s t
test.
Figure S4. Silencing miR408 by the artificial microRNA technique. (a) Schematic diagram
of the amiR408 expression vector. The vector pAth-miR319a contains the Arabidopsis primiRNA sequence of miR319a and is used as a template for PCR. Six primers (pA, pB, and
pI–pIV) are required to replace both the endogenous miR319a and miR319a* sequences with
miR408 and amiR408* sequences. The resultant PCR product is directionally cloned into
pJIM19 at the XhoI and SpeI sites. 35Sp, cauliflower mosaic virus 35S promoter; Tnos,
nopaline synthase terminator; Bar, Basta resistance gene. (b) Quantitative RT-PCR analysis
of pre-miR408 and mature miR408 levels in seedlings of four independent amiR408
transgenic lines. The amiR408-1 line was chosen for further experiments shown in Figure 4
of the main text. Actin7 and 5S rRNA were used as the endogenous controls, respectively. (c)
RNA blot analysis of miR408 accumulation in the seedlings of wild type and individual
amiR408 lines. U6 snRNA was used as the loading control. (d) qRT-PCR analysis of miR408
target genes of Plantacyanin and LAC13 in wild type and individual amiR408 transgenic
plants. Error bars represent SD (n = 3). (e) Expression levels of At2g47020 in the amiR408
lines as determined by qRT-PCR using both primer pairs as indicated in Figure S3.
Figure S5. Complementation of the FLAG T-DNA mutants by constitutive activation of
miR408.
(a) RNA blot analysis of miR408 levels in wild type and the two T-DNA mutants expressing
the 35S:miR408 transgene. For each T-DNA mutant, two independent rescue lines expressing
35S:miR408 were chosen for analysis. U6 snRNA was used as loading control. (b) qRT-PCR
analysis of the miR408 target genes Plantacyanin and LAC13 in wild type, the two miR408
T-DNA mutants, and the rescue lines. Actin7 was used as an endogenous control. Error bars
represent SD (n = 3). (c) Morphological comparison of wild type, miR408 T-DNA mutants,
and rescue lines at the seedling and adult stages. Bars = 1 cm. (d) Quantitative analysis of
fresh weight, root length, leaf length and width. Fresh weight and root length were measured
with 12-d-old seedlings grown on MS medium. Leaf length and width were measured with
the fully expanded fifth rosette leaf of adult plant grown in soil. Data are mean ± SD (n ≥ 15).
* p < 0.01 or ** p < 0.001 vs wild type by Student’s t test.
Table S1. List of primers in this study.