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%.