Figure Legends
Figure 1. Yield-related traits of Koshihikari and Takanari rice
cultivars under control and chronic O3 conditions.
(A) Total dry weight, (B) grain yield, (C) harvest index, (D) 1000-seeds
weight, (E) total grain number, and (F) panicle number were measured in
Koshihikari (red) and Takanari (blue) under control and chronic
O3 conditions in 2019 and 2020. The results are shown as
relative values (%) obtained under chronic O3conditions to those obtained under control conditions. * and ** indicate
significant differences of each trait under control and chronic
O3 conditions in Koshihikari and Takanari at p< 0.05 and 0.01, respectively (n = 4–11). The dashed line
represents the line where the value for each trait is 100%.
Figure 2. Photosynthesis-related parameters and plant
appearance of Koshihikari and Takanari rice cultivars under control and
chronic O3 conditions in 2019.
(A) CO2 assimilation rate (A ), (B) stomatal
conductance (g s), (C) intercellular
CO2 concentration (C i), and (D)
electron transport rate for photosystem Ⅱ (ETRⅡ) per unit leaf area were
measured in fully expanded leaves of Koshihikari (red) and Takanari
(blue) at 49–50, 68–69, 88–89, and 108–109 days after the beginning
of treatment (DAT) in 2019. The measurements were conducted at a
CO2 concentration of 400 µmol mol-1, a
PPFD of 1500 µmol m–2 s–1, and an
air temperature of 30°C. The results are shown as relative values (%)
obtained under chronic O3 conditions (pale color) to
those obtained under control (deep color) conditions (n = 4–6 for each
condition). In addition, (E) A was measured under a series of
CO2 concentrations of 100, 200, 300, 400, 500, 600, 750,
900, 1200 and 1500 µmol mol–1 at 54–56 DAT in 2019.
(F) The maximum rate of ribulose-1,5-bisphosphate (RuBP) carboxylation
(V cmax) was estimated. The pictures of the plant
appearance of (G) Koshihikari (red) and (H) Takanari (blue) under
control (deep color) and chronic O3 (pale color)
conditions were taken at 53 DAT in 2019. In addition, the SPAD value was
measured on the top 1st, 2nd,
3rd, and 4th leaves (I) Koshihikari
and (J) Takanari plants at 49–50 DAT in 2019. Vertical and horizontal
bars in each panel indicate the standard error (n = 4–5). * and **
indicate significant differences of each parameter under control and
chronic O3 conditions in Koshihikari and Takanari atp < 0.05 and 0.01, respectively. The dashed line
represents the line where the value for each trait is 100%. The values
in each column in panel (F) represent the relative value under chronic
O3 conditions to that under control conditions.
Figure 3. Leaf biochemical and morphological traits in
Koshihikari and Takanari rice cultivars under control and chronic
O3 conditions in 2019.
(A) Nitrogen content, (B) chlorophylla +b content, (C) RuBisCO content, (D) RuBisCO activation state, (E) stomatal
density, and (F) guard cell length were measured for Koshihikari (red)
and Takanari (blue) on the same leaf as gas exchange measurements were
conducted in 2019. The results are shown as relative values (%)
obtained under chronic O3 conditions to those obtained
under control conditions (n = 4–6 for each condition). * and **
indicate significant differences of each trait under control and chronic
O3 conditions in Koshihikari and Takanari at p< 0.05 and 0.01, respectively. The dashed line represents the
line where the value for each trait is 100%.
Figure 4. Photosynthesis and growth-related traits of
Koshihikari and Takanari rice cultivars under control and chronic
O3 conditions in 2020.
(A) CO2 assimilation rate per unit leaf area (A )
was measured in fully expanded leaves of Koshihikari (red) and Takanari
(blue) at 66–67 and 89–90 days after the beginning of treatment (DAT)
in 2020. In addition, we estimated (C) the CO2assimilation rate at single-leaf level (A leaf) by
multiplying (B) a single leaf area and A . At 89–90 DAT, (D)
plant height, (E) tiller number, (F) total dry weight, (G) whole
leaf-blade area and (H) weight, and (I) the ratio of the weights of
whole leaf blades to leaf sheaths were measured. The results are shown
as relative values (%) obtained under chronic O3conditions to those obtained under control conditions. ** indicates
significant differences of each trait under control and chronic
O3 conditions in Koshihikari and Takanari at p< 0.05 and 0.01, respectively (n = 4–5). The dashed line
represents the line where the value for each trait is 100%.
Figure 5. Correlation analysis among photosynthesis, growth,
and yield-related traits under control and chronic O3conditions of Koshihikari and Takanari rice cultivars.
The correlation co-efficients of the CO2 assimilation
rate per unit leaf area (A ), stomatal conductance
(g s), intercellular CO2concentration (C i), electron transport rate
(ETR), CO2 assimilation rate at the single-leaf level
(A leaf), single leaf-blade area (LA), plant
height (PH), tiller number (TN), grain weight (GW), and total dry weight
(TDW) were analyzed. In addition, we plotted the replication data of GY
against (C, E) LA and (D, F) TN observed under control (deep color) and
chronic O3 (pale color) conditions in (C, D) Koshihikari
(red) and (E, F) Takanari (blue) plants. * and ** indicate significant
correlations among parameters at p < 0.05 and 0.01,
respectively.
Figure 6. Differentially expressed genes and enriched
biological processes under control and chronic O3conditions of Koshihikari and Takanari rice cultivars.
Differentially expressed genes (DEGs) with (A) up- and (B)
downregulation under chronic O3 conditions were detected
in Koshihikari and Takanari at 66–67 and 89–90 days after the
beginning of treatment (DAT) in 2020. The DEG intersections among the
four groups of the combination of two cultivars at two growth stages
were visualized by connecting groups with plots. The numbers in the
columns represent the number of DEGs for each intersection. The
positively (C, E, G, I) and negatively (D, F, H, J) enriched GO terms
associated with biological process with the top-10 lowestp -values are shown for (C–F) Koshihikari and (G–J) Takanari at
66–67 and 89–90 DAT, respectively. Horizontal columns represent the
number of DEGs included in enriched GO terms.
Figure 7. Co-expression network of differentially expressed
genes in Koshihikari rice cultivar under control and chronic conditions.
(A) Gene co-expression network was analyzed in the differentially
expressed genes (DEGs) under chronic O3 conditions. The
module name and its gene number were listed with top-15 highest
correlation co-efficient (R ) values between module eigengene and
single leaf area (SLA). In addition, the detailed network was visualized
for the (B) brown, (C) yellow, (D) blue, and (E) turquoise modules. Each
node represents a gene, and the shade and size of the node reflect the
values of degree and Betweenness centrality, respectively. Each edge
represents the connection between nodes. The square nodes are the genes
that were detected as hub-gene candidates in each module.
Figure 8. Co-expression network of differentially expressed
genes in Takanari rice cultivar under control and chronic conditions.
(A) Gene co-expression network was analyzed in the differentially
expressed genes (DEGs) under chronic O3 conditions. The
module name and its gene number were listed with top-15 highest
correlation co-efficient (R ) values between module eigengene and
tiller number (TN). In addition, the detailed network was visualized for
the (B) brown, (C) yellow, (D) blue, and (E) turquoise modules. Each
node represents a gene, and the shade and size of the node reflect the
values of degree and Betweenness centrality, respectively. Each edge
represents the connection between nodes. The square nodes are the genes
that were detected as hub-gene candidates in each module.
Figure 9. Gene co-expression network of green module in
Koshihikari rice cultivar under chronic O3 conditions
(A) The detailed network was visualized for the green module shown in
Fig. 7. Each node represents a gene, and the shade and size of the node
reflect the values of degree and Betweenness centrality, respectively.
Each edge represents the connection between nodes, and the width of
edges reflects the weight values. The square nodes are the genes that
were detected as hub-gene candidates in the module. In addition, the
expression level of the three hub-gene candidates, (B)Os01g0662700 , (C) Os03g0563300 , and (D)Os12g0291100 , was compared in Koshihikari (red) and Takanari
(blue) under control and chronic O3 conditions at 66-67
and 89-90 DAT. The results are shown as relative values (%) of the
values obtained under chronic O3 conditions to those
obtained under control conditions (n = 4–5). The dashed line represents
the line where the value for each trait is 100%.