1. Volume 7, Issue 2, Pages 404-421 (February 2014)
SALT-RESPONSIVE ERF1 Is a Negative Regulator of Grain Filling and Gibberellin- Mediated Seedling Establishment in Rice 
Romy Schmidt, Jos H.M. Schippers, Delphine Mieulet, Mutsumi Watanabe, Rainer Hoefgen, Emmanuel Guiderdoni, Bernd Mueller- Roeber 
Molecular Plant 
Volume 7, Issue 2, Pages (February 2014)
DOI: /mp/sst131
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 SERF1 Expression during Grain Development.
(A–H) GUS activity staining of transgenic plants harboring the SERF1:GUS construct. (A, B) SERF1 expression is detected in stamens of young spikelets while, at later stages before flowering (C, D), the expression of SERF1 is detected mainly in the vascular bundles of palea and lemma. (E) Additionally, before flowering and during grain filling, SERF1 expression is observed in the rachis (arrow). (F) In a single branch of a panicle, GUS staining correlates with the developmental status of spikelets; that is, those that have progressed and produced an immature seed show no staining (arrows) while those that did not still show strong GUS staining. (G, H) After pollination, the expression of SERF1 declines in the vascular bundles of the palea and lemma and gets confined to the base of the developing grain (arrows indicate GUS staining).
(I) Relative expression level of SERF1 during grain filling as determined by qRT–PCR. RNA was extracted from immature wild-type seeds at 0, 4, 8, and 15 DAF. Expression levels are given on a log scale expressed as 40 – ΔCT ± SE. 40 equals the expression level of the 17S-rRNA reference gene that was used for normalization; n = 3. DAF, days after flowering.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Effect of SERF1 on Heading and Panicle-Related Traits.
(A) Panicle emergence and (B) flowering time of serf1 (T5 generation) and SERF1 knockdown (KD) lines (T3 generation) compared to WT and EV controls.
(C) Panicle emergence in Ubi:SERF1 (OE) and 35S:SERF1–CFP (COE) plants (T0 generation).
(D) Phenotypic comparison between WT, serf1, and SERF1 knockdown (KD) lines at the heading stage.
Panicle number of (E) WT, serf1, and SERF1 knockdown (KD) and (F) EV, Ubi:SERF1 (OE), and 35S:SERF1–CFP (COE) plants.
Panicle length of (G) WT, serf1, and SERF1 knockdown (KD) and (H) EV, Ubi:SERF1 (OE), and 35S:SERF1–CFP (COE) plants.
(I) Number of spikelets per panicle and (J) grains per panicle of serf1 and SERF1 knockdown (KD) plants (% of control).
(K) Delayed flowering of Ubi:SERF1 (OE) and 35S:SERF1–CFP (COE) plants as compared to EV plants.
(L) Grain number per plant of serf1 and SERF1 knockdown (KD) lines given as percentage of WT (serf1) or EV (KD lines). For analysis of panicle emergence, flowering time, and panicle-related traits, 15 wild-type plants were compared with 15 serf1 and SERF1 knockdown plants, respectively, and 12 EV plants with 12 Ubi:SERF1 and 35S:SERF1–CFP plants. Asterisk (*) indicates a significant difference (p ≤ 0.05) to the corresponding control as determined by Student’s t-test. WT, wild type; EV, empty vector.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
SERF1 Expression Is Negatively Correlated with Grain Size and Starch Content.
(A) Phenotypic comparison of wild-type (1), serf1 (2), and SERF1 knockdown grains, KD 3-1 (3), KD 4-1 (4), and KD 5-1 (5).
(B) Phenotypic comparison of grains of EV (1), SERF1 overexpression lines OE 14-1 (2), OE 27-1 (3), and lines expressing 35S:SERF1–CFP, namely COE 1-1 (4), COE 14-1 (5), and COE 15-1 (6).
(C) Length, width, and thickness of serf1 and SERF1 knockdown grains (% of control). n = 50.
(D) Grain weight of serf1 and KD grains relative to wild-type and EV grains, respectively. Grains were measured in triplicate (300 seeds each).
(E) Length, width, and thickness of OE and COE grains relative to EV grains. n = 50.
(F) Grain weight of OE and COE grains relative to EV grains. Grain weight was measured in triplicate (80 seeds each).
(G) Starch (%) of wild-type, serf1, and KD grains. Five biological replicates with six grains each were used for analysis.
(H) Swelling of wild-type and serf1 endospermal starch after incubation with urea (0–6 M) for 24 h. Data in graphs represent means ± SE. Asterisk (*) indicates a significant difference (p ≤ 0.05) to the corresponding control calculated by Student’s t-test. WT, wild-type; EV, empty vector.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Impact of SERF1 Knockout on RPBF, Starch Biosynthesis, and SSP Gene Expression during Grain Development.
Seeds of serf1 and wild-type were harvested at 0, 4, 8, and 15 DAF. In addition to RICE PROLAMIN-BOX BINDING FACTOR (RPBF) and RICE SEED B-ZIPPER 1 (RISBZ1), starch biosynthesis genes (GBSSI, GRANULE-BOUND STARCH SYNTHASE I; SSI, SSIIa, SSIIIa, STARCH SYNTHASE I, IIa, IIIa; AGPL2, ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2; AGPS2b, ADP-GLUCOSE PYROPHOSPHORYLASE SMALL SUBUNIT2b; PPDKB, PYRUVATE ORTHOPHOSPHATE DIKINASE B), SSP genes (Glu, glutelin; GluA, GluB, GluD; Glb-1, globulin 1; Prol14, prolamin 14; RP10, 10-kDa prolamin; RM1, 13-kDa prolamin; RAG-1, seed allergenic protein) and aleurone layer number-related genes (OsCR4, CRINKLY 4; OsDEK1, DEFECTIVE KERNEL 1; OsSAL1, SUPERNUMERARY ALEURONE LAYER 1) were measured. Expression levels are given on a log scale expressed as 40 – ΔCT ± SE. 40 equals the expression level of the 17S-rRNA reference gene that was used for normalization. n = 3. Asterisk (*) indicates a significant difference (p ≤ 0.05) to expression in wild-type seeds calculated by Student’s t-test. DAF, days after flowering.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
RPBF and Starch Biosynthesis Gene Expression in Immature Seeds of SERF1 Overexpression Plants.
Seeds of OE 14-1 and EV were harvested at 0 and 4 DAF. In addition to RPBF, the starch biosynthesis genes GBSSI, SSI, SSIIa, SSIIIa, AGPL2, AGPS2b, and PPDKB were measured. Expression levels are given on a log scale expressed as 40 – ΔCT ± SE. 40 equals the expression level of the 17S-rRNA reference gene that was used for normalization. n = 3. Asterisk (*) indicates a significant difference (p ≤ 0.05) to expression in EV seeds calculated by Student’s t-test. EV, empty vector; DAF, days after flowering.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
SERF1 Directly Regulates the Expression of RPBF and GBSSI by Binding to a DREB-Specific Cis-Element.
(A) Positions of the putative SERF1 binding sites ‘ACCGAC’ (white box), ‘GCCGAC’ (black box), and ‘GTCGAC’ (gray box) in the promoters of RPBF and GBSSI. In addition, positions and lengths of probes used for EMSA (blue arrows) and primers used for ChIP–qPCR (black arrows) are shown.
(B) Relative luciferase activity (FC) of RPBF:LUC and GBSSI:LUC in the presence of SERF1 or GAD–SERF1, respectively, in rice leaf protoplasts. n = 3. An asterisk (*) indicates a significant difference (p ≤ 0.05) to the basal activity (control) calculated by Student’s t-test.
(C) EMSA performed with probes specific to RPBF (left panel) and GBSSI (right) as given in (A). Binding of SERF1 causes a band shift (arrows), which disappears upon competition (‘Comp.’) with unlabelled probe (100-fold molar excess).
(D) Consensus sequence of the SERF1 binding site.
(E) ChIP–qPCR assay revealed binding of SERF1 to the promoters of RPBF (motif R1R2) and GBSSI (motifs G1 and G3). Mean enrichment of three biological replicates ± SE is presented. NC1 and NC2 represent negative controls for which a RPBF promoter fragment approximately 1.6 kb and a GBSSI promoter fragment approximately 1.2 kb upstream of the TSS, respectively, were amplified. Asterisks (*) indicate a significant difference (p ≤ 0.05) to the negative IgG control calculated by Student’s t-test. FC, fold change.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7 SERF1 Negatively Affects Germination and Seedling Growth.
(A) Expression (FC, fold change) of RAmy1A, RPBF, and GAMyb in roots of 6-day-old serf1, KD 4-1, and OE 27-1 seedlings relative to wild type. n = 3.
(B) Starch clearing zone assay with embryo-less half-seeds of wild-type and serf1 plants incubated on agar plates (0.1% starch) without and in the presence of 1 μM GA3. Starch was stained with iodine solution. Cleared zones indicate starch degradation.
(C) Growth phenotype of 6-day-old WT, EV, serf1, and SERF1 knockdown (KD 3-1, KD 4-1, and KD 5-1) seedlings grown under control conditions.
(D) Germinated seeds (%) of serf1, KD 4-1, and WT under control conditions at 1–7 d after sowing (DAS). n = 36.
(E) Root and shoot lengths of serf1 and WT seedlings 3–7 DAS. n = 32.
(F) Germinated seeds (%) of EV and SERF1 overexpression (OE 14-1, OE 27-1) lines under control conditions at 1–7 DAS. n = 32.
(G) Growth phenotype of 7-day-old EV, OE 14-1, and OE 27-1 seedlings grown under control conditions.
(H) Root and shoot lengths of EV, OE 14-1, and OE 27-1 seedlings at 4 and 7 DAS. n = 32. Asterisk (*) indicates significant difference (p ≤ 0.05) to wild-type and EV seedlings, respectively, calculated by Student’s t-test. WT, wild type; EV, empty vector.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Loss of SERF1 Increases Resistance to the GA Biosynthesis Inhibitor Paclobutrazol.
(A) Inhibitory effect of paclobutrazol (PAC; 2 μM) on shoot and root growth of 7-day-old wild-type, serf1, and SERF1 knockdown (KD) and (B) EV and SERF1 overexpression (OE) seedlings. n = 32. Data represent means ± SE. Length is given relative to mock-treated seedlings of the same genotype. Asterisk (*) in (A) and (B) indicates significant difference (p ≤ 0.05) to treated wild-type or EV seedlings, respectively, calculated by Student’s t-test.
(C) Expression analysis of GA biosynthesis/signaling genes in roots and (D) shoots of 6-day-old serf1 and SERF1 knockdown (KD 4-1) seedlings. Heatmaps represent expression (log2FC) relative to either wild-type (for serf1 mutant) or EV seedlings (for KD 4-1), respectively, according to the given color code.
(E) Induction of SERF1 expression (FC) in roots of 6-day-old wild-type seedlings exposed to 50 μM GA3 for 2h as compared to mock-treated seedlings. For (C)–(E), three independent biological replicates (five plants each) were analyzed.
(F) Growth response of 7-day-old serf1 seedlings to 1 μM ABA and (G) 10 μM 1-aminocyclopropane-1-carboxylic acid (ACC) application. Shoot and root lengths are given relative to mock-treated seedlings. n = 32. WT, wild type; EV, empty vector; FC, fold change.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
Model of SERF1 Action on RPBF and GBSSI during Deve lopment and Germination of Rice Seeds.
RPBF is a positive co-regulator of GBSSI expression during grain filling and RAmy1A expression during germination by binding to the prolamin-box in the respective promoters in a GA-dependent manner. During both developmental processes, the action of RPBF is limited by SERF1 which binds to the DREB-element in its promoter and represses expression of the TF gene. Additionally, SERF1 down-regulates GBSSI activity during the early stages of grain development through directly interacting with its promoter. In the depicted model, SERF1 fine-tunes GA-dependent developmental processes by acting as a molecular brake on the positive regulator RPBF.
Molecular Plant 2014 7, DOI: ( /mp/sst131)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions