Favorable Alleles of GRAIN-FILLING RATE1 Increase the Grain-Filling Rate and Yield of Rice

Abstract

Hybrid rice (Oryza sativa) has been cultivated commercially for 42 years in China. However, poor grain filling still limits the development of hybrid japonica rice. We report here the map-based cloning and characterization of the GRAIN-FILLING RATE1 (GFR1) gene present at a major-effect quantitative trait locus. We elucidated and confirmed the function of GFR1 via genetic complementation experiments and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing in combination with genetic and molecular biological analyses. In addition, we conducted haplotype association analysis to mine the elite alleles of GFR1 among 117 rice accessions. We observed that GFR1 was constitutively expressed and encoded a membrane-localized protein. The allele of the rice accession Ludao (GFR1 ^Ludao) improved the grain-filling rate of rice by increasing Rubisco initial activity in the Calvin cycle. Moreover, the increased expression of the cell wall invertase gene OsCIN1 in the near isogenic line NIL-GFR1 ^Ludao promoted the unloading of Suc during the rice grain-filling stage. A yeast two-hybrid assay indicated that the Rubisco small subunit interacts with GFR1, possibly in the regulation of the rice grain-filling rate. Evaluation of the grain-filling rate and grain yield of F1 plants harboring GFR1 ^Ludao and the alleles of 20 hybrids widely cultivated commercially confirmed that favorable alleles of GFR1 can be used to further improve the grain-filling rate of hybrid japonica rice.

Figure 1.

Phenotypic analysis of grain filling among Ludao, NIL-GFR1^Ludao, and C-bao. A, Plants at the grain-filling stage. This is a composite image with three photographs. Bars = 10 cm. B, Seed shape after maturation. Bar = 1 cm. C to E, Morphological changes in brown rice grains of Ludao (C), NIL-GFR1^Ludao (D), and C-bao (E) at five stages. Bars = 1 cm. F, Brown rice weight changes of Ludao, NIL-GFR1^Ludao, and C-bao at five stages. G, Grain-filling rate changes of Ludao, NIL-GFR1^Ludao, and C-bao at five stages. H, Comparison of yield traits between C-bao and NIL-GFR1^Ludao in 2011. The data are means ± sd (n = 3). Student’s t test was used for statistical analysis (*, P ≤ 0.05 and **, P ≤ 0.01).

Figure 2.

Map-based cloning and identification of GFR1. A, GFR1 was finely mapped onto the long arm of chromosome 10. Molecular markers and numbers of recombinants are labeled above and below the filled bars, respectively. The candidate ORF1 (LOC_Os10g36400) is marked in red. The mutation sites of ORF1 are also shown. ATG and TGA represent the start and stop codons, respectively. B, Complementation identification of GFR1: morphological changes of brown rice grains of C-bao and C16 of the GFR1^C-bao line (T3 transgenic plants) at five stages. Bar = 1 cm. C, Overexpression identification of GFR1: morphological changes of brown rice grains of Nipponbare and OE-GFR1^Nipponbare (T3 transgenic plants) at five stages. Bar = 1 cm. D, Grain-filling rate changes in C-bao and GFR1^C-bao. E, Grain-filling rate changes in Nipponbare and OE-28 of the OE-GFR1^Nipponbare line. F, Brown rice weight changes of C-bao and GFR1^C-bao. G, Brown rice weight changes of Nipponbare and OE-GFR1^Nipponbare. H, Grain size of C-bao and GFR1^C-bao. I, Grain size of Nipponbare and OE-GFR1^Nipponbare. J, Initial Rubisco activity of C-bao and GFR1^C-bao. K, Initial Rubisco activity of Nipponbare and OE-GFR1^Nipponbare. The data are means ± sd (n = 3). Student’s t test was used for statistical analysis (*, P ≤ 0.05 and **, P ≤ 0.01).

Figure 3.

Knocking out GFR1 in Nipponbare decreases the grain-filling rate and grain weight. A, T-DNA expression construct for Cas9 and gRNA for the GFR1 gene in rice. The Cas9 nuclease is under the control of the doubled 35S promoter, and transcription was terminated by the nos terminator. The rice U6 promoter (OsU6) was used to drive the expression of the single guide RNAs (sgRNAs). LB, Left border; RB, right border. B, Sequence comparisons of the targeting site of the KO plants generated by CRISPR-Cas9 technology. The two sgRNA target sequences are underlined in blue and green. WT, Wild type. C, Comparison of the brown rice weight and grain-filling rate changes of Nipponbare and KO plants at five stages. The data are means ± sd (n = 3). D, Morphological changes in the brown rice grain of Nipponbare and KO plants at the five grain-filling stages. Bar = 1 cm.

Figure 4.

Analysis of the expression of GFR1. A, Relative expression levels of GFR1 are shown in the tissues of C-bao and NIL-GFR1^Ludao, including grains and flag leaves at five grain-filling stages, and in stems, roots, stamens, and stigmas. The 18S rRNA gene was used as an internal control. The data are means ± sd (n = 3). Student’s t test was used for statistical analysis (*, P ≤ 0.05 and **, P ≤ 0.01). B to J, Promoter activity analysis of GFR1 in NIL-GFR1^Ludao. B, Root. Bar = 1 cm. C, Center of flag leaf. Bar = 1 cm. D, Stem. Bar = 1 cm. E, Transverse section of a stem. Bar = 1 cm. F, Young panicle. Bar = 1 cm. G, Spikelet. Bar = 5 mm. H, Stamen. Bar = 5 mm. I, Pistil. Bar = 1 mm. J, Brown rice grains at different DAF. Bar = 5 mm.

Figure 5.

Characterization of the GFR1 protein in rice. A, Phylogenetic tree of GFR1 in Arabidopsis, rice, and other monocotyledonous species. The phylogenetic tree was constructed using MEGA 5.0. Bootstrap values are shown as percentages at the nodes. The scale bar at the bottom represents the genetic distance. B to E, Subcellular localization of the GFR1-GFP fusion protein. Membrane localization of the GFR1-GFP fusion protein (B), the location of the chloroplast (C), a merged image of the bright field and the chloroplast (D), and a merged image (E) are shown. Bars = 50 μm. F, Y2H assay showing that GFR1 interacts with proteins of the cDNA library. DDO, Control medium (SD/-Trp-Leu); QDO, selective medium (SD/-Ade/-His/-Leu/-Trp/Aba). G, Transactivation activity assays of GFR1. H, Y2H assay showing that GFR1 of C-bao and Ludao interacts with OsRbcS. I, Y2H assay showing that GFR1 of C-bao and Ludao interacts with OsRbcL. AD, Activation domain; BD, binding domain; SD, synthetic dropout. J, Glutathione S-transferase (GST) pull-down assay of GFR1 and OsRbcS. GFR1 expressed as part of a GST fusion protein was pulled down by His-OsRbcS.

Figure 6.

Microscopy assay of the endosperm between C-bao and NIL-GFR1^Ludao. A and B, Transmission electron microscopy images of the central part of endosperm cells in C-bao. C to E, Scanning electron microscopy images of cross sections of seeds of C-bao. F and G, Transmission electron microscopy images of the central part of endosperm cells in NIL-GFR1^Ludao. H to J, Scanning electron microscopy images of cross sections of seeds of NIL-GFR1^Ludao. K to V, Paraffin sections of grains of C-bao (K, L, O, P, S, and T) and NIL-GFR1^Ludao (M, N, Q, R, U, and V) at 3, 7, and 14 DAF, respectively. L, P, T, N, R, and V show magnifications of the dashed boxes in K, O, S, M, Q, and U, respectively. a, Amyloplast; A, Aleurone layer; Es, Endosperm; n, nucleus; Pe, pericarp.

Figure 7.

Transcriptome profiling in flag leaves and grains at 14 DAF of C-bao and NIL-GFR1^Ludao. A, MA plot of DEGs in flag leaves and grains at 14 DAF of C-bao and NIL-GFR1^Ludao. The number next to the arrow indicates the number of DEGs. FPKM, Fragments per kilobase of exon model per million mapped fragments. MA, M versus A. M is minus and A is add. B, Overview of DEGs in flag leaves and grains at 14 DAF of C-bao and NIL-GFR1^Ludao. C, DEGs related to the GFR according to the KEGG pathway. The color in each cell indicates the value of the log₂ fold change (NIL-GFR1^Ludao/C-bao).

Figure 8.

Hypothetical model of the function of GFR1 in terms of the grain-filling rate. PGA, 3-phosphoglycerate; GAP, glyceralgehyde-3-phosphate; RuBP, ribulose-1,5-bisphosphate.