Supporting Information Legends
Figure S1. HPLC chromatograms of glutenin and the relative proportions of glutenin fractions (inserted figures)
in mature wheat grains grown under four combinations of N and S supply. The four treatments were NS (3 mM
N and 2 mM S), NS- (3 mM N and 0.02 mM S), N+S- (15 mM N and 0.02 mM S), and N+S-/N+S (shift at mid-grain
filling from N+S- to 15 mM N and 2 mM S). The peaks corresponding to HMW (Dy12, By9, Dx2, Bx7, and Ax2*)
and LMW (LMW6,7, a, b, c, etc.) glutenin subunits are indicated.
Figure S2. HPLC chromatograms of gliadin and the relative proportions of gliadin fractions (inserts) in mature
wheat grains grown under four combinations of N and S supply. The four treatments were NS (3 mM N and 2
mM S), NS- (3 mM N and 0.02 mM S), N+S- (15 mM N and 0.02 mM S), and N+S-/N+S (shift at mid-grain filling
from N+S- to 15 mM N and 2 mM S). The peaks corresponding to ω5-, ω1,2-, α/β-, and γ-gliadin are indicated.
Figure S3. Gene expression and rate of accumulation of LMW-GS (a, b, c) and HMW-GS (d, e, f) versus thermal
time after anthesis for wheat grains grown under three combinations of N and S supply. The three treatments
were NS- (3 mM N and 0.02 mM S), N+S- (15 mM N and 0.02 mM S), and NS (3 mM N and 2 mM S). Data are
means ± 1 s.e. for n = 2 biological replicates for gene expression and for n = 3 for the rate of protein
accumulation.
Figure S4. Gene expression and rate of accumulation of gliadin fractions versus thermal time after anthesis for
wheat grains grown under three combinations of N and S supply. The three treatments were NS- (3 mM N and
0.02 mM S), N+S- (15 mM N and 0.02 mM S), and NS (3 mM N and 2 mM S). (a), (b), (c) for ω5-gliadin; (d), (e), (f)
for ω1,2-gliadin; (g), (h), (i) for α/β-gliadin; (j), (k), (l) for γ-gliadin. Data are means ± 1 s.e. for n = 2 biological
replicates for gene expression and for n = 3 for the rate of protein accumulation.
Figure S5. Mass of free amino acids per grain versus thermal time after anthesis for wheat grains grown under
four combinations of N and S supply. The four treatments were NS (3 mM N and 2 mM S), NS- (3 mM N and 0.02
mM S), N+S- (15 mM N and 0.02 mM S), and N+S-/N+S (shift at mid-grain filling from N+S- to 15 mM N and 2
mM S). Data are means ± 1 s.e. for n = 3 biological replicates.
Figure S6. Mass of primary metabolites versus thermal time after anthesis for wheat grains grown under four
combinations of N and S supply. The four treatments were NS (3 mM N and 2 mM S), NS- (3 mM N and 0.02 mM
S), N+S- (15 mM N and 0.02 mM S), and N+S-/N+S (shift at mid-grain filling from N+S- to 15 mM N and 2 mM S).
GSSG, glutathione disulfide. GSH, glutathione. Data are means ± 1 s.e. for n = 3 biological replicates.
Figure S7. Correlation of gene expression quantifications by microarray and qPCR for wheat grains grown under
four combinations of N and S supply. The y-axis labels are probes of the same genes amplified on x-axis. The
four treatments were NS (3 mM N and 2 mM S), NS- (3 mM N and 0.02 mM S), N+S- (15 mM N and 0.02 mM S),
and N+S-/N+S (shift at mid-grain filling from N+S- to 15 mM N and 2 mM S). The Pearson's correlation
coefficient r and the P value are indicated in each panel.
Figure S8. Heat map of the 122 differential expression of genes (listed in Table S4) in the four nitrogen (N) and
sulfur (S) treatments. Data were scaled and log2-transformed and are represented in a color scale with purple
for higher expression and green for lower expression. The average expression levels of each cluster is shown in
the right panels. NS, black solid line; NS-, red line; N+S-, blue line, N+S-/N+S, blue dashed line.
Table S1. Dry mass, N mass and concentration, S mass and concentration, and protein composition of mature wheat
grains grown with four combinations of N and S supply. The four treatments were NS (3 mM N and 2 mM S), NS- (3 mM
N and 0.02 mM S), N+S- (15 mM N and 0.02 mM S), and N+S-/N+S (shift at mid-grain filling from N+S- to 15 mM N
and 2 mM S). Data are means ± 1 s.e. for n = 3 independent replicates. Different letters within a row indicate significant
differences (α = 0.05) among N/S treatments following a Tukey test. P-values from ANOVAs for the four N/S treatments
are reported.
Table S2. Estimated rate and duration of the accumulation of grain dry mass, total N, total S, and grain
storage proteins. Data were fitted with a 3‐parameter logistic function equation.Where Q is the quantity of dry
mass or N per grain, t is the degree‐days after anthesis, Qmax is the final value of Q approached as t → ∞, r is
the maximum rate of accumulation defined as the derivative of the point of inflexion, and t95 is the duration of
accumulation defined as the period from anthesis onwards in which 95% of Qmax is accumulated.
Table S3. Summary statistics of linear regressions between the rate of accumulation of grain
storage protein subunits or classes and expression of their respective genes in developing wheat
grains grown under three combinations of N and S supply. Slopes and intercepts are based on
standard major axis regressions. CI, confidence intervals. Different letters indicate statistical
differences (α = 0.05) in the intercept or slope. Data are plotted in Figure 5.
Table S4. Clusters and functional annotations of the 122 genes differentially affected by N and S supply. The probe ID o
S8) is provided following its putative functional category and functional annotation. Name abbreviations are provided f
abbreviations were used in the directed multi-level regulatory network for node name (Figure 8). Cluster no. indicates t
heatmap (Figure S8), and Module no. indicates the network module in which a gene is involved in the network (Figure
transcription factor. CM, chloroform methanol-soluble protein. AA, amino acid.