PS content is reduced in <i>sui1-4</i> plants.

<p>(A) and (B) Measurement of PS and its putative substrate PE. Values are means ± standard error of three independent experiments. (*0.01</p

<i>sui1-4</i> is a secretion disorder mutant.

<p>(A) Fractions of membrane proteins extracted from 2-week-old WT and <i>sui1-4</i> plants immunoblotted with anti-OsCESA antibody. Anti-H⁺ATPase and anti-Arf1 were two marker proteins predominantly present in the plasma membrane and endomembrane fractions, respectively. The dextran (DEX) fraction contains endomembranes; the polyethylene glycol (PEG) fraction contains PMs. (C) Immunoblotting of secreted green fluorescent protein (secGFP) in WT and <i>sui1-4</i> protoplasts in a transient expression system. Equal amounts of protein extracted from protoplasts and culture medium were detected using anti-cFBPase, a marker for the plant cytosol. Immunoblotting was conducted in triplicate. 0, 1, 3, 10 (ug) represent the amounts of plasmid (SecGFP) when transient expression using rice protoplast. (B) and (D) Quantification of protein secretion in (A) and (C) relative to total protein (defined as 100%) using ImageJ. Three independent experiments were performed to obtain the average secretory efficiency. Differences were significant (**p<0.01, Student’s <i>t</i>-test). Error bars indicate standard deviation.</p

Phenotype of the <i>shortened uppermost internode 1–4 (sui1-4)</i> mutant.

<p>(A) Phenotype of the wild type (WT) plant and <i>sui1-4</i> mutant at the heading stage. Scale bar: 10 cm. (B) Internode lengths of the WT (left) and <i>sui1-4</i> (right) plants. Internode length was the distance between two adjacent nodes (white arrowheads). Inset: magnification of red rectangular section in (B), the uppermost internode (indicated by brackets) of <i>sui1-4</i> plants was shortened (Inset scale bar: 2 mm). Scale bar: 10 cm. (C) to (F) Internode lengths in WT and <i>sui1-4</i> plants. Differences were significant for each internode (**p<0.01, Student’s <i>t</i>-test). Error bars indicate standard deviation. (G) and (H) Longitudinal sections of the second internode of WT and <i>sui1-4</i> plants. Scale bars: 100 μm. (I) and (J) Cell length and cell width calculated from measurements of longitudinal sections of the second internode of WT and <i>sui1-4</i> plants. (K) and (L) Transverse sections of the second internode of WT and <i>sui1-4</i> plants. Scale bars: 100 μm. (M) Radii of stems calculated from measurements of transverse sections of the second internode of WT and <i>sui1-4</i> plants. Data are means ± standard error (n = 10). **p<0.01. (N) Comparison of cell numbers of radii in transverse sections of the second internode of WT and <i>sui1-4</i> plants (n = 10). NS, not significant.</p

Monosaccharide composition of cell walls from various organs.

<p>Monosaccharide composition of cell walls from various organs.</p

<i>OsPSS-1</i> expression and histochemical staining of <i>pOsPSS-1</i>::<i>GUS</i> transgenic plants.

<p>(A) qRT-PCR shows that <i>OsPSS-1</i> is ubiquitously expressed in various organs, with the highest level in panicles. For each organ, the <i>sui1-4</i> mutant displayed lower expression levels than WT plants. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s <i>t</i>-test (**p<0.01). (B) to (F) Histochemical staining of young seedlings one week after germination (B), at four weeks (C), one week before heading (D), and at the heading stage (E). Signals were detected in roots and coleoptiles (B), in young panicles and the basal regions of internodes (C) and (D), and divisional and elongating zones of the uppermost internode (E). The images in (E) and (F) are from two parts of the same transgenic plant. Scale bars: 1 cm in (B) to (D), 5 cm in (E) and (F). (G) Enlargement of histochemical staining boxed in (B). Scale bar: 100 μm. (H) Enlargement of hand-cut transverse section of the stem boxed in (E). Scale bar: 500 μm.</p

Subcellular localization of OsPSS-1-GFP 36 h after transformation in <i>Arabidopsis</i> protoplasts.

<p>(A) to (F) Confocal micrographs of the distributions of OsPSS-1-GFP (green) and indicated markers (magenta) 36 h after transformation. ER, endoplasmic reticulum; PM, plasma membrane; PVC, prevacuolar compartment; TGN/EE, trans-Golgi network/early endosomes. Scale bars: 10 μm. (G) For quantification, the PSC coefficients shown in the right panel (r_p) were calculated after analysis of at least 10 individual protoplasts. The level of colocalization ranges from +1 for perfect correlation to 1 for negative correlation.</p

Subcellular localization of OsPSS-1-GFP 12 h after transformation in <i>Arabidopsis</i> protoplasts.

<p>(A) Immunoblot of proteins isolated from rice protoplasts expressing OsPSS-1-GFP. CM, cell membrane; CS, cell soluble; P, pellet; S, supernatant. (B) to (G) Confocal micrographs of the distributions of OsPSS-1-GFP (green) and indicated markers (magenta) 12 h after transformation. ER, endoplasmic reticulum; PM, plasma membrane; PVC, prevacuolar compartment; TGN/EE, trans-Golgi network/early endosomes. Scale bars: 10 μm. (H) For quantification, the PSC coefficients shown in the right panel (r_p) were calculated after analysis of at least 10 individual protoplasts. The level of colocalization ranges from +1 for perfect correlation to -1 for negative correlation.</p

Subcellular localization of GFP-LactC2 12 h after transformation in <i>Arabidopsis</i> protoplasts.

<p>(A) to (F) Confocal micrographs of the distributions of GFP-LactC2 and indicated markers 12 h after transformation. ER, endoplasmic reticulum; PM, plasma membrane; PVC, prevacuolar compartment; TGN/EE, trans-Golgi network/early endosomes. Scale bars: 10 μm. (G) For quantification, the PSC coefficients shown in the right panel (r_p) were calculated after analysis of at least 10 individual protoplasts. The level of colocalization ranges from +1 for perfect correlation to 1 for negative correlation.</p

Positional cloning of <i>OsPSS-1</i>.

<p>(A) Fine mapping of the <i>OsPSS-1</i> locus. Molecular markers and number of recombinants (recs) are shown. BAC, bacterial artificial chromosome; ORF, open reading frame. (B) and (C) The WT genomic segment of <i>OsPSS-1</i> completely rescues plant stature. L1 and L2 denote plants from T1 transgenic lines. In (C), the nodes of the <i>sui1-4</i> mutant and L1 plants are indicated with white arrowheads. Scale bars: 10 cm. (D) <i>OsPSS-1-</i>RNAi transgenic lines mimic the phenotype of <i>sui1-4</i> plants. R1, R2, and R3 represent three independent T1 transgenic lines. Scale bar: 10 cm. (E) Internodes in WT, R1, R2, and R3 plants; white arrowheads indicate the nodes. Scale bar: 10 cm. (F) qRT-PCR reveals lower <i>OsPSS-1</i> expression in mutant plants than in WT plants, but transcript levels of OsPSS-1 homologs (LOC_Os05g48060 and LOC_Os01g49024) were not affected. Values are means ± standard error of three independent experiments. Significant differences were identified with Student’s <i>t</i>-test (**p<0.01); NS, not significant.</p

<em>Ehd4</em> Encodes a Novel and <em>Oryza</em>-Genus-Specific Regulator of Photoperiodic Flowering in Rice

<div><p>Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (<i>Oryza sativa</i> L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the <i>CO</i>-<i>FT</i> module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (<i>Hd1</i>-<i>Hd3a</i> in rice). However, in rice, there is a unique <i>Ehd1</i>-dependent flowering pathway that is <i>Hd1</i>-independent. Here, we report isolation and characterization of a positive regulator of <i>Ehd1</i>, <i>Early heading date 4</i> (<i>Ehd4</i>). <i>ehd4</i> mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that <i>Ehd4</i> encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids <i>in vitro</i> and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. <i>Ehd4</i> expression is most active in young leaves with a diurnal expression pattern similar to that of <i>Ehd1</i> under both short-day and long-day conditions. We show that <i>Ehd4</i> up-regulates the expression of the “florigen” genes <i>Hd3a</i> and <i>RFT1</i> through <i>Ehd1,</i> but it acts independently of other known <i>Ehd1</i> regulators. Strikingly, <i>Ehd4</i> is highly conserved in the <i>Oryza</i> genus including wild and cultivated rice, but has no homologs in other species, suggesting that <i>Ehd4</i> is originated along with the diversification of the <i>Oryza</i> genus from the grass family during evolution. We conclude that <i>Ehd4</i> is a novel <i>Oryza</i>-genus-specific regulator of <i>Ehd1</i>, and it plays an essential role in photoperiodic control of flowering time in rice.</p> </div