Supporting Information Legends
Figure S1. Tissue expression pattern of OsZIP3 in basal node and node I.
(a,b) Enlarged vascular bundles (a) and remaining tissues (RT) (b) of the basal node
regions separated by laser microdissection.
(c-e) Phloem (EVBP) (c) and xylem (EVBX) (d) regions of enlarged vascular bundles
and diffuse vascular bundles (DVB) (e) of node I separated by laser microdissection.
(f,g) Tissue-dependent expression of OsZIP3 in basal node (f) and node I (g). The
expression level was determined by quantitative RT-PCR. Expressions relative to RT (f)
and EVBP (g) are shown.
HistoneH3 (f) or Actin (g) was used as an internal control.
Data are means ±SD of three biological replicates.
Figure S2. Response of OsZIP3 protein and mRNA to Zn deficiency or excess in the
basal node.
(a,b) Response of OsZIP3 protein level to Zn deficiency (a) or excess (b) in the basal
node. Immunostaining was performed by using basal node of seedlings exposed to the
solution without Zn (-Zn) for 6 d or to 10 µM Zn for 48 hrs. Bar = 100 µm.
(c) Response of OsZIP3 mRNA to different Zn concentrations. Rice was cultivated in a
nutrient solution with 0, 0.4 or 10 µM for 3 days. The expression level was determined
by quantitative real-time RT-PCR. Expression relative to 0.4 µM Zn is shown.
Histone
H3 was used as an internal control. Data are means ±SD of three biological replicates.
Figure S3. Zn concentration in different organs.
After both the wild-type rice and RNAi lines were cultivated in a one-half strength
Kimura B solution for 20 days, different organs were separately harvested. Zn was
determined with ICP-MS after digestion. Data are means ±SD of three biological
replicates. One biological replicate was used two plants. Statistical comparison was
performed by one-way ANOVA followed by the Tukey’s multiple comparison test. All
data were compared with the wild type (*P < 0.05, **P < 0.01).
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Figure S4. Concentration of Zn, Mn, Cu and Fe in different organs at the reproductive
growth stage.
(a-d) Concentration of Zn (a), Mn (b), Cu (c) and Fe (d) in different organs at the
reproductive growth stage. Both wild-type rice and RNAi lines were cultivated in
one-half strength Kimura B solution until heading stage.
Different organs were
separately harvested and subjected to determination of metals by ICP-MS. Data are
means ±SD of three biological replicates. One biological replicate was used two plants.
Statistical comparison was performed by one-way ANOVA followed by the Tukey’s
multiple comparison test. All data were compared with the wild type (*P < 0.05, **P <
0.01).
Figure S5. Comparison of OsHMA2 expression in the wild-type rice and OsZIP3
knockdown lines.
(a-c) The expression level of OsHMA2 in the wild type and knockdown lines.
After the seedlings of both wild-type rice and three OsZIP3 RNAi lines were grown in
an one-half strength Kimura B solution for 26 d, the roots (a), shoot basal regions (b),
and shoots (c) were sampled for determination of OsHMA2 expression with RT-PCR.
Expression relative to the wild-type rice (a-c) is shown.
HistoneH3 and Actin was
used as internal controls. Data are means ±SD of three biological replicates. Statistical
comparison was performed by one-way ANOVA followed by the Tukey’s multiple
comparison test. All data were compared with the wild type (*P < 0.05).
Figure S6. Ct value of HistoneH3 in different organs.
(a) The Ct value of HistoneH3 in different organs at different growth stages. Rice was
grown in a paddy field till ripening and different organs were sampled.
(b) The Ct value of HistoneH3 to metal deficiency in the shoot basal regions.
Rice
was cultivated in a nutrient solution with or without Zn, Fe, Mn or Cu for one week.
The expression level was determined by quantitative real-time RT-PCR. Data are means
±SD of three biological replicates.
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