OsELF3 Is Involved in Circadian Clock Regulation for Promoting Flowering under Long- Day Conditions in Rice  Ying Yang, Qiang Peng, Guo-Xing Chen, Xiang-Hua Li, Chang-Yin Wu  Molecular Plant  Volume 6, Issue 1, Pages 202-215 (January 2013) DOI: 10.1093/mp/sss062 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phylogenetic Tree of the ELF3 Family Proteins. The alignment for each gene family dataset was used for distance-based phylogenetic analysis with the MEGA4.0 (Kumar et al., 2008) software packages. Twenty-nine ELF3 homologs were selected for establishing a bootstrap N-J phylogenetic tree and 1000 replicates were conducted to determine the statistical support for each node. Smo, Selaginella moellendorffii; Mtr, Medicago truncatula; Gma, Glycine max; Vvi, Vitis vinifera; Zma, Zea mays; Sbi, Sorghum bicolor; Bdi, Brachypodium distachyon; Osa, Oryza sativa; Ppa, Physcomitrella patens; Rco, Ricinus communis; Mes, Manihot esculenta; Csa, Cucumis sativus; Aly, Arabidopsis lyrata; Ath, Arabidopsis thaliana; Cpa, Carica papaya; Mgu, Mimulus guttatus. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of OsELF3 and Its Mutants. (A) Structure of OsELF3 and Tos17 insertion site. Four exons (filled boxes) and three introns (lines between the filled boxes) are shown. Tos17 was inserted into the second intron as the open triangle presents. Arrows indicate the primers used for analyzing the insertion site, and open boxes indicate the 5′ and 3′ UTRs. (B) Genotyping of OsELF3 segregates with primers: M, homozygous for Tos17 insertion; W, wild-type; H, heterozygous. Primers P1 and P2 flanking the Tos17 insertion site amplify a product from heterozygous or WT allele. PCR-positive plants with P2 and P3 indicate Tos17 insertion in the examined site. Presence of a product with P2 and P3 and not with P1 and P2 indicates a plant homozygous. (C) The phenotype-rescued oself3 plant by genetic complementation (positive) and oself3 mutant transformed by empty pCAMBIA2301 vector (control). (D, E) Phenotypes of WT (left) and oself3 (right) at heading stage. Plants were cultivated in a growth room under LDs (14h light/10h dark, 28°C) (D) and SDs (10h light/14h dark, 28°C) (E), respectively. Photographs were taken at 65d after soaking. (F) Days to heading in WT and oself3 mutant plants under LDs or SDs. Days to heading was scored when first panicle bolted after soaking. Error bars indicate standard deviations; n=15 plants. (G, H) Comparison of leaf emergence rates between WT plants and oself3 mutants under LDs (G) and SDs (H) during development (means±SD, n=15). Leaf emergence rate was calculated according to the measurements described by Itoh et al. (1998). Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Gene Structure and Phenotype of ubi::OsELF3 Plants. (A) Overexpression construct for rice transformation. Hpt, Hygromicin phosphotransferase; LB, left border; NOS, NOS terminator; RB, right border; TEVL, TEV leading signal sequence; Ubi, maize ubiquitin promoter. (B) Heading date of two OsELF3 overexpression lines (OX-3 and OX-6) compared with control in SDs (14h light/10h dark, 25°C) and LDs (10h light/14h dark, 25°C), respectively. Days to heading was scored when the first panicle bolted after planting. ZH11 transformed negative (left) was as control. Error bars indicate standard deviations; n=10 plants, ** P<0.01. (C) Phenotypes of OsELF3 overexpressing plant (OX) and control at heading stage. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Diurnal Expression Patterns of OsELF3, OsGI, Hd1, Ehd1, RFT1, and Hd3a in WT (Filled Dark Circles) and oself3 (Open Gray Circles) Plants in SDs and LDs by qRT–PCR Analysis. The expression levels are relative to the UBQ mRNA. In all panels, the mean of each point is based on the average of three biological repeats calculated using the relative quantification method (Livak and Schmittgen, 2001). ZT, Zeitgeber time. ZT=0 is defined as the time of light on. The open and filled bars at the bottom represent the light and dark periods, respectively. Error bars, s.e.m. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Diurnal Expression Patterns of OsLHY, OsPRR1, OsPRR95, OsPRR73, and OsPRR37 in WT (Filled Dark Circles) and oself3 (Open Gray Circles) Plants in SDs and LDs by qRT–PCR Analysis. The expression levels are relative to the UBQ mRNA. In all panels, the mean of each point is based on the average of three biological repeats calculated using the relative quantification method (Livak and Schmittgen, 2001). ZT, Zeitgeber time. ZT=0 is defined as the time of light on. The open and filled bars at the bottom represent the light and dark periods, respectively. Error bars, s.e.m. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Free-Running Rhythms of OsELF3 and OsLHY mRNA Expression in Continuous Light Conditions. WT (filled dark circles) and oself3 (open gray circles) seedlings were entrained under LDs (16h light/8h dark, 28°C) conditions and then shifted to constant light (28°C) at time 24, which corresponds to normal dawn. Samples were taken every 4h and the expression levels are relative to the UBQ mRNA. In all panels, the mean of each point is based on the average of three biological repeats calculated using the relative quantification method (Livak and Schmittgen, 2001). The open and ashy bars at the bottom represent the light and subjective dark periods, respectively. The numbers below the bars indicate the time in gathering the samples from the beginning (0). Error bars, s.e.m. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 A Model for OsELF3-Mediated Photoperiodic Control of Flowering by Regulation of the Circadian Clock in Rice under LDs. The circadian clock-related genes constitute a series of interconnected negative and positive feedback loops. The OsELF3 may directly repress the expression of some members of the OsPRRs family. OsELF3 may participate in the degradation of OsGI and repress the expression of Ghd7. Molecular Plant 2013 6, 202-215DOI: (10.1093/mp/sss062) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions