Huang ZH , Bao K , Jing ZH , Wang Q , Duan HF , Zhang S , Tao WW , Wu QN .

	FIGURE 1. Seed comparison of North Gordon Euryale (WT) and Hybrid Euryale (HL). After harvest in September (20th week), Phenotype (A), length (B), width (C), ratio of width to length (D), weight (E), and hundred-seed weight (F) of fresh seeds of the WT and HL were detected. Bar = 1 cm. Data are mean ± SD. Two-tail Student’s t-test was performed between the WT and HL (*p < 0.05; **p < 0.01; ***p < 0.001).
	FIGURE 2. Metabolic profiling analysis of the WT and HL. (A) KEGG classification of significantly different contents in dry mature seeds (harvested in the 20th week) between the WT and HL. Each point represents the degree of enrichment of the KEGG entry, and the size of each point indicates the number of different metabolites enriched in the KEGG pathway. (B) Heat map of metabolites with significantly different contents. Every square block indicates a metabolite, and significantly up-regulated and down-regulated metabolites are displayed in red and blue, respectively. HL, hybrid Euryale lines; WT, wild type (North Gordon Euryale).
	FIGURE 3. Histological analysis of the ovary of WT and HL fruits. (A) Longitudinal sections (left, Scale bar = 5 mm) and representative paraffin sections of the reproductive organizations of 12-week-old WT and HL (right, Scale bar = 1 mm). The ovary, denoted with the dotted box, was larger in HL fruits when compared with WT at the same developmental stage. (B) Cells around the ovary of WT and HL were observed using a fluorescent microscope. Images including bright field and red autofluorescence are shown. Scale bar = 100 μm. (C) Cell length of WT and HL were evaluated using six 12-week-old fruits per line. Statistical count was shown as the frequency distribution of the cell length. HL, hybrid Euryale lines; WT, wild type (North Gordon Euryale).
	FIGURE 4. Classification and auxin response of EuSAUR62. (A) Phylogenetic relationships of EuSAUR62 and SAURs from Arabidopsis thaliana and Oryza sativa. SAURs from Arabidopsis and rice are displayed in red and cyan, respectively. EuSAUR62 (black) is next to AtSAUR62 and AtSAUR75. (B) The subcellular localization of EuSAUR62 in tobacco leaves. EuSAUR62 was fused to GFP and transfected into tobacco leaves. A merged image including bright field, H2B-mCherry and GFP fluorescence was observed using a confocal laser scanning microscope (Scale bar = 40 μm). (C) Relative expression of EuSAUR62 in the fruits of WT and HL was detected using qRT-PCR. qRT-PCR analyses of EuSAUR62 expression in 2-week-old WT seedlings after treating with different doses of exogenous IAA (0, 1, 10, 50 μM) for 1 h (D) or with 10 μM exogenous IAA from 0 to 60 min, followed by replacing the IAA solution with pure water (E). The time points of IAA treatment are shown in orange columns, whereas water culturing is expressed in blue columns. Euryale Actin was used as an internal control. Relative expression of EuSAUR62 in WT seedlings following 1 h treatment of 0, 10, 100, or 500 μM exogenous NPA (F), an auxin transport inhibitor, or with 10 μM exogenous NPA treatment for 1 h before water culturing (G). The time points of NPA treatment are shown in crimson columns, whereas water culturing is expressed in blue columns. Two-tail Student’s t-test was used for statistical analysis; Data are mean ± SD. *p < 0.05. HL, hybrid Euryale lines; IAA, Indole-3-acetic acid; NPA, N-1-naphthylphthalamic acid; WT, wild type (North Gordon Euryale).
	FIGURE 5. Grain size and histology comparison of the WT and OE-EuSAUR62 rice. After harvest, (A) morphologies of the mature grains produced by OE-EuSAUR62 and WT were observed. The grains generated by the OE-EuSAUR62 were significantly longer than that of WT. Scale bar = 1 cm. Histological analysis of the glumes (B) and outer glume cells (C) at heading stage. The glumes of WT and transformant were observed using a stereo microscope (Scale bar = 4 mm). The outer glume cells were observed under a scanning electron microscope (Scale bar = 100 μm). After harvest, 10 grains per line were collected randomly for size and weight measurement. OE-EuSAUR62 significantly improved the grains in weight (D) and length (E) compared with the WT. The glume length of OE-EuSAUR62 rice was longer than the WT (F). Length of the outer glume cells was longer in OE-EuSAUR62 rice compared to the WT (G). Statistical count is shown as the frequency distribution of the cell length. (H) Localization of IAA in the rice ovary (white arrow) of WT and OE-EuSAUR62 at the flowering stage (Scale bar = 500 μm). (I) The expression of EuSAUR62 was detected using western blot analysis with an anti-GFP antibody. The β-actin was used as an internal control. The negative controls are represented in the Supplementary material. Data are mean ± SD. One-way ANOVA and post hoc statistical analysis was performed (**p < 0.01, ***p < 0.001). OE-EuSAUR62, EuSAUR62 overexpression lines; WT, wild type rice.
	FIGURE 6. Interaction of EuSAUR62 and OsPIN9. (A) EuSAUR62 and OsPIN9 were fused with the corresponding YFP parts for BiFC assay in tobacco leaves. Images including bright field, H2B-mCherry and YFP fluorescence were observed using a laser confocal microscope (Scale bar = 20 μm). (B) Membrane yeast two-hybrid assay of the interaction of EuSAUR62 and OsPIN9. Interactions were determined based on cell growth on SD-T-L-H with 3-AT. IAA, Indole-3-acetic acid; OE-EuSAUR62, EuSAUR62 overexpression lines; WT, wild type rice. (C) Tobacco leaves were transfected with constructs containing a p35S:OsPIN9-GFP and/or p35S:EuSAUR62-Flag. Proteins were immunoprecipitated with anti-GFP antibodies and subjected to western blot analysis with anti-Flag antibodies to confirm the presence of the OsPIN9 protein and to anti-GFP gel-blot analysis to reveal the EuSAUR62 partner.
	FIGURE 7. EubZIP55 activates expression of EuSAUR62. (A) The subcellular localization of EubZIP54/55 in tobacco leaves. EubZIP54/55 were fused to GFP and transfected into tobacco leaves. Merged images including bright field, H2B-mCherry, and GFP fluorescence were observed using a confocal laser scanning microscope. Relative expression of EubZIP54 (B) and EubZIP55 (C) in the fruits of WT and HL was detected using qRT-PCR. (D) Yeast one-hybrid assay for the DNA-protein interaction between the EuSAUR62pro and EubZIP54 or EubZIP55. The indicated constructs were transformed into yeast reporter strain Y187 and grown on SD–L-T-H media with different concentrations of 3-AT. pGADm53 vs. pHIS-p53 was used as a positive control, and pGADT7 vs. pHIS-EuSAUR62pro was used as a negative control. Schematic design is shown on the left (E). Transcription repression and activation activity of EubZIP54 (F) and EubZIP55 (G) on EuSAUR62, respectively, which is expressed by the ratio of firefly luciferase to Renilla luciferase (LUC/REN). (H) GUS activity of EuSAUR62pro and 35S promoter was compared in tobacco leaves (Scale bar = 2 mm). p35S:GUS was used as a positive control, and empty:GUS was used as a negative control. Two-tail Student’s t-test was used for statistical analysis; Data are mean ± SD. *p < 0.05, **p < 0.01. EuSAUR62pro, EuSAUR62 promoter; HL, hybrid Euryale lines; WT, wild type (North Gordon Euryale).
	FIGURE 8. Functional characterization of EubZIP55 on the auxin-mediated apical hook of Euryale seedlings. Relative expression of EubZIP55 (A) and EuSAUR62 (B) in seedlings of WT and HL under constant light or dark conditions. (C) Phenotypes of apical curvature in the 2-week-old seedlings of WT and HL under constant light or dark conditions. The sun represents constant light treatment, and the moon represents dark treatment. Curvature degree is shown as a perpendicular line in the upper right of each picture. Scale bar = 2 cm. (D) The quantitative assessment of apical curvature of seedlings. Two-tail Student’s t-test was used for statistical analysis; Data are mean ± SD. **p < 0.01, ***p < 0.001.
	FIGURE 9. A working model of the role of EuSAUR62 in regulating Euryale seed size. In HL, light increases the expression of EubZIP55, contributing to the higher expression level of EuSAUR62, which regulates the localization of auxin by interacting with PINs, thereby promoting ovary enlargement and increasing seed size. In the WT, free-localization of auxin on the exocarp weakens the regulation of seed development and probably promotes the development of pricks.

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