The Rice HGW Gene Encodes a Ubiquitin-Associated (UBA) Domain Protein That Regulates Heading Date and Grain Weight

Heading date and grain weight are two determining agronomic traits of crop yield. To date, molecular factors controlling both heading date and grain weight have not been identified. Here we report the isolation of a hemizygous mutation, heading and grain weight (hgw), which delays heading and reduces grain weight in rice. Analysis of hgw mutant phenotypes indicate that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea, both composing the spikelet hull that is known to determine the size and shape of brown grain. Molecular cloning and characterization of the HGW gene showed that it encodes a novel plant-specific ubiquitin-associated (UBA) domain protein localized in the cytoplasm and nucleus, and functions as a key upstream regulator to promote expressions of heading date- and grain weight-related genes. Moreover, co-expression analysis in rice and Arabidopsis indicated that HGW and its Arabidopsis homolog are co-expressed with genes encoding various components of ubiquitination machinery, implying a fundamental role for the ubiquitination pathway in heading date and grain weight control

Dwarf Tiller1, a Wuschel-related homeobox transcription factor, is required for tiller growth in rice.

Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that Dwarf Tiller1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a Wuschel-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice

qRT-PCR expression analysis of heading- and grain weight-related genes in WT and <i>hgw</i> mutant.

<p>(A) to (D). Expression of heading-related genes including <i>Ehd1</i> (A), <i>Hd1</i> (B), <i>Hd3a</i> (C) <i>and OsGI</i> (D) in panicles collected from 3 time points. 1: 11:00, 2: 16:00 and 3: 20:00. (E). Expression of grain weight-related genes including <i>GIF1</i>, <i>GW2</i>, <i>GW5</i> and <i>GS3</i> in panicles of WT and <i>hgw</i> mutant. The transcript levels of examined genes were normalized to the <i>UBQ1</i> expression levels. All values are based on at least three biological and three technical repeats and presented as means ± SE (n≥3).</p

Cloning of the <i>HGW</i> gene.

<p>(A). Exon/intron structure of the <i>HGW</i> gene and T-DNA insertion site. Five exons (filled boxes) and four introns (lines between the filled boxes) are shown. T-DNA was inserted into the first exon. Arrows indicate primers used for analyzing the insertion site. LB and RB represent the left and right borders of T-DNA. (B). PCR genotyping of T1 generation plants. PCR positive bands indicate insertion of the T-DNA enhance trap casette in the rice genome (P4+P5) or in the first exon of <i>LOC_Os06g06530</i> (P2+P3), whereas PCR positive bands obtained with P1 and P2 primers suggest presence of undisrupted WT <i>LOC_Os06g06530</i> gene. (C). Phenotypic complementation of the <i>hgw</i> mutant by the <i>LOC_Os06g06530</i> gene. (D). Grain phenotypes of the complemented lines and the WT control. (E). Days to heading of the WT control and complemented plants. (F). Grain width of 20 seeds of the WT control and complemented plants. (G). Weight of 1000 brown grains of the WT control and complemented plants. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034231#s2" target="_blank">Results</a> are presented as means ± SE (n = ≥9). Control in C to G: WT; Com in C to G: complemented plants.</p

Expression analysis of <i>HGW</i> in WT and <i>hgw</i> mutant.

<p>(A). RT-PCR analysis of <i>HGW</i> in 11 tissues, including roots at seedling stage with two tillers (R1), leaves at two-tiller stage (L1), roots at 4–5-cm young panicle stage (R2), leaves at 4–5-cm young panicle stage (L2), sheath when young panicle was at secondary branch primordial differentiation stage (Sh), stem at 5 days before heading (St) and panicle at 5 different development stages (P1 to P5 were amplified from panicle with sizes of 0.5, 1, 1.5–2, 3–3.5, 6.5 cm, respectively). The expression level of a <i>GAPDH</i> gene was used as an internal control. (B). qRT-PCR analysis of <i>HGW</i> in 10 tissues as indicated. The expression level of <i>UBQ1</i> was used as an internal control. (C). qRT-PCR analysis of <i>HGW</i> in 4 tissues of WT (Control) and <i>hgw</i> at maturity. The expression level of a ubiquitin gene was used as an internal control. All data are presented as means ± SE (n≥3).</p

Expression pattern of <i>HGW</i> indicated by GUS staining in different tissues of the <i>hgw</i> mutant.

<p>GUS staining was observed in ligule (A), leaf blade (B), sheath (C), culm (D and E), spikelet (F and G) and grains (H). (E) shows GUS staining in the cross-section of culm, and (G) reveals GUS staining in stamen and pistil.</p

Histological analyses of spikelet hulls at maturity.

<p>(A). Spikelet hulls of the WT control and <i>hgw</i> mutant. Dotted lines indicate positions of cross-sections. (B). Scanning electron micrographs showing cross-sections of the WT control and <i>hgw</i> mutant. The yellow line outlines the overall circumference of the outer parenchyma cell layer. the blue line outlines lemma and the red lines outlines palea in WT control or <i>hgw</i> mutants. (C). Magnified view of spikelet hull cross-section boxed in B (left). (D). Magnified view of spikelet hull cross-section boxed in B (right). (E). Circumferences of the outer parenchyma cell layers of WT and <i>hgw</i> panicle (overall, outlined with yellow line), lemma (outlined with blue line) and palea (outlined with red line). (F). Cell numbers of lemma and palea of WT and <i>hgw</i>. (G). Cell size of lemma and palea of WT and <i>hgw</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034231#s2" target="_blank">Results</a> of E to G were obtained from cross-sections and are presented as means ± SE (n = 3).</p

Phenotypic analysis of <i>hgw</i>.

<p>(A) and (B). Grain phenotypes of <i>hgw</i> and the WT control. (C). Grain width of 20 seeds of <i>hgw</i> and the WT control. (D). Grain length of 10 seeds of <i>hgw</i> and the WT control. (E). Weight of 1000 brown grains of <i>hgw</i> and the WT control. (F). Days to heading of <i>hgw</i> and the WT control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034231#s2" target="_blank">Results</a> are presented as means ± SE (n≥9). (G) and (H). Phenotypes of <i>hgw</i> and the WT control at maturity.</p

Subcellular localization of HGW protein during transient expression in rice protoplast cells.

<p>(A). A rice protoplast cell expressing HGW::YFP-HGW (green). Chloroplasts in the cell were visualized by chlorophyll autofluorescence (red). (B). A rice protoplast cell expressing HGW::YFP-HGW (green) and 35S::RFP (red). (C). A rice protoplast cell expressing HGW::HGW-YFP (green). Chloroplasts in the cell were visualized by chlorophyll autofluorescence (red). (D). A rice protoplast cell expressing HGW::HGW-YFP (green) and 35S::RFP (red). (E). A rice protoplast cell expressing HGW::YFP-HGW (green) and stained with the Hoechst 33342 nuclear dye (Blue). (F). A rice protoplast cell expressing HGW::HGW-YFP (green) and stained with the Hoechst 33342 nuclear dye (Blue). Nomarski DIC and merged images of the protoplasts are presented. The sizes of cells are indicated by the sizes of scale bars.</p