Figure 1.
(A) and (B). Grain phenotypes of hgw and the WT control. (C). Grain width of 20 seeds of hgw and the WT control. (D). Grain length of 10 seeds of hgw and the WT control. (E). Weight of 1000 brown grains of hgw and the WT control. (F). Days to heading of hgw and the WT control. Results are presented as means ± SE (n≥9). (G) and (H). Phenotypes of hgw and the WT control at maturity.
Figure 2.
Histological analyses of spikelet hulls at maturity.
(A). Spikelet hulls of the WT control and hgw mutant. Dotted lines indicate positions of cross-sections. (B). Scanning electron micrographs showing cross-sections of the WT control and hgw 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 hgw 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 hgw 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 hgw. (G). Cell size of lemma and palea of WT and hgw. Results of E to G were obtained from cross-sections and are presented as means ± SE (n = 3).
Figure 3.
(A). Exon/intron structure of the HGW 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 LOC_Os06g06530 (P2+P3), whereas PCR positive bands obtained with P1 and P2 primers suggest presence of undisrupted WT LOC_Os06g06530 gene. (C). Phenotypic complementation of the hgw mutant by the LOC_Os06g06530 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. Results are presented as means ± SE (n = ≥9). Control in C to G: WT; Com in C to G: complemented plants.
Figure 4.
Sequence analysis of proteins encoded by HGW and its homologs.
(A). Sequence alignment of proteins encoded by HGW and its homologs, generated with the CLUSTALX program. The UBA domain is outlined with the red box. (B). WebLogo [28] of the most conserved consensus motifs in the UBA domains, obtained from http://expasy.org/cgi-bin/prosite/sequence_logo.cgi?ac=PS50030. Stack heights represent conservation at a position, and symbol heights within a stack represent the relative frequency of each residue.
Figure 5.
Subcellular localization of HGW protein during transient expression in rice protoplast cells.
(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.
Figure 6.
Expression analysis of HGW in WT and hgw mutant.
(A). RT-PCR analysis of HGW 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 GAPDH gene was used as an internal control. (B). qRT-PCR analysis of HGW in 10 tissues as indicated. The expression level of UBQ1 was used as an internal control. (C). qRT-PCR analysis of HGW in 4 tissues of WT (Control) and hgw at maturity. The expression level of a ubiquitin gene was used as an internal control. All data are presented as means ± SE (n≥3).
Figure 7.
Expression pattern of HGW indicated by GUS staining in different tissues of the hgw mutant.
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.
Figure 8.
qRT-PCR expression analysis of heading- and grain weight-related genes in WT and hgw mutant.
(A) to (D). Expression of heading-related genes including Ehd1 (A), Hd1 (B), Hd3a (C) and OsGI (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 GIF1, GW2, GW5 and GS3 in panicles of WT and hgw mutant. The transcript levels of examined genes were normalized to the UBQ1 expression levels. All values are based on at least three biological and three technical repeats and presented as means ± SE (n≥3).