A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality

Abstract

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Figure 1.

Phenotype of the flo2 Mutant. (A) Representative seed of the wild type (Kinmaze), the flo2 mutant (EM37), vector control line (VC), and complemented line (CL). CL and VC are transformants with/without a wild-type FLO2 gene in the flo2 mutant EM37. The top panel shows the grain shapes of the seeds, and the bottom panel shows seeds illuminated with backlight. The floury phenotype (EM37 and VC) is indicated by a dark image. Wild-type and CL grains were more packed and transmitted light, whereas the flo2 mutant and VC grains were chalky and floury, resulting in dark images when illuminated with backlight. Bar = 1 cm. (B) Scanning electron microscopy images of the transverse sections of the wild-type, flo2 mutant, VC, and CL grains. Bars = 50 μm.

Figure 2.

Amylose Content and Amylopectin Composition of the flo2 and Wild-Type Grain. (A) Amylose content of the wild type (Kinmaze), flo2 mutant (EM37), VC, and CL. Representative VC and CL were analyzed. Error bars show sd (n = 6). (B) to (D) Comparison of the chain-length profile of amylopectins. Debranched amylopectin was compared with the wild type. Differences from the wild type in chain length distribution of amylopectins are shown. Amylopectin profile of the flo2 grain (B), CL (C), and VC (D).

Figure 3.

Expression Levels of the Genes Involved in Production of Storage Starch and Protein in the flo2 Mutant. Total RNA extracted from 10 DAF developing seeds was used for real-time RT-PCR analysis. Expression of representative genes involved in storage starch production, storage proteins, and carbon metabolism in the flo2 mutant (EM37) are shown relative to the wild type (Kinmaze), which is set as 1. Each gene name is indicated by a simplified representation with the accession number of the corresponding full-length cDNA. BE1, BEIIa, and BEIIb, branching enzyme I, IIa, and IIb, respectively; AGPL (AGPL1, AGPL2, AGPL3, and AGPL4) and AGPS (AGPS1, AGPS2a, and AGPS2b), ADP glucose pyrophosphorylase large subunit and small subunit, respectively; SSI, SSIIa, SSIIb, SSIIc, SSIIIa, SSIIIb, SSIIIc, SSIVa, and SSIVb, soluble starch synthase I, IIa, IIb, IIc, IIIa, IIIb, IIIc, IVa, and IVb, respectively; GBSSI and GBSSII, granule-bound starch synthase I and II; ISA1, ISA2, and ISA3, corresponding isoamylase isozymes; PUL, pullulanase; Susy1, Susy2, and Susy3, sucrose synthase 1, 2, and 3, respectively; PPDKB, pyruvate phosphate dikinase B; PGI-a and PGI-b, glucose-6-phosphate isomerase a and b; Amy3B, Amy3C, Amy3D, and Amy3E, α-amylase 3B, 3C, 3D, and 3E, respectively; GluA1, GluA2, GluA3, GluB1, and GluB4, glutelin A1, A2, A3, B1, and B4, respectively; Globulin1, Globulin2, 11S-globulin, and 19-kD globulin, corresponding globulin species; 10-, 13-, and 17-kD prolamin, prolamins with each size; RA16, RA17, RAG5B, RAG2, and RG21, species of rice allergenic proteins; AlaAT, alanine aminotransferase; PDI, protein disulfate isomerase. Asterisks show subunits of the plastidic AGPase. Error bar shows sd (n = 3).

Figure 4.

Accumulation of Glutelins, BEI, and RA16 in Developing Seeds. (A) Accumulation of glutelins in developing seeds of the wild type (Kinmaze), flo2 mutant (EM37), VC, and CL. Each lane contains proteins from one-hundredth of the total glutelin fraction, which was extracted from the mixture of 10 rice grains of each line. Proteins are shown by Coomassie blue staining after separation on SDS-PAGE. Arrows indicate 51-kD glutelin precursor, 30- to 36-kD α-subunits of glutelins, and 19- to 22-kD β-subunits of glutelins, respectively. Size markers are shown on the left. (B) and (C) Accumulation of BEI (B) and RA16 (C) in developing seeds 10 DAF of the wild type, flo2 mutant, VC (three independent lines), and CL (five independent lines) is shown by protein gel blot analysis using antiserum raised against BEI and RA16, respectively. The RA16 antiserum detects highly conserved 14- to 16-kD allergenic proteins with slightly different molecular mass. Each lane contains 1 μg of total protein, which was separated by SDS-PAGE.

Figure 5.

Positional Cloning and Structure of Rice FLO2. (A) Fine mapping of the FLO2 locus. The region of the FLO2 locus was mapped to a 354-kb region by published EST/SSR markers (RM3814 and RM3335-1), then narrowed to a 37-kb region by newly created markers (218042 and 218787) in chromosome 4 (Chr. 4), which contained four predicted genes shown by the gene names registered in the database. Markers used for the mapping are shown in the figure and are detailed in Supplemental Table 2 online. (B) Exon/intron structure of FLO2. The FLO2 gene is composed of 23 exons (filled box) with 22 introns, including three TPR motifs (open box) in the middle. Eight mutants showing the flo2 phenotype contained one base substitution in the FLO2 gene that generated a premature stop codon (EM36, EM37, EM139, EM280, and EM554), a presumable splicing site (EM373 and EM624), or an amino acid change (EM756).

Figure 6.

Phylogenetic Tree of the Representative FLO2 Homologs. Proteins are shown in the figure as the names of plant species with the corresponding accession numbers registered in protein databases (http://www.ncbi.nlm.nih.gov/ and http://www.ddbj.nig.ac.jp/index-j.html). Subfamilies including FLO2, FLL1, and FLL2 are indicated. Rice genes are boxed. Scale represents the number of differences between sequences.

Figure 7.

Interaction of LEA and bHLH with FLO2. (A) and (B) Y2H analysis of FLO2 with a LEA protein (accession number AK061818) (A) and a bHLH protein (accession number AK070651) (B). LEA, bHLH, FLO2(N), and FLO2(C) indicate yeast cells containing plasmids for expression of the LEA, bHLH, FLO2_(1–898), and FLO2_(900–1720), respectively. EV(B) and EV(P) show those containing the empty bait and prey vectors instead of the corresponding genes, respectively. (C) and (D) Reciprocal swapping using LEA and FLO2 (C) and bHLH and FLO2 (D). FLO2(W) and FLO2(M) indicate yeast cells containing plasmids for expression of the full-length FLO2 and FLO2_(535–1189), respectively. These cells were grown on agar plates lacking Trp, Leu, and His in the medium.

Figure 8.

Detection of the FLO2–bHLH Interaction by an in Vitro Pull-Down Experiment. Proteins on SDS-PAGE were detected by Coomassie blue staining. Lanes 1 to 5 indicate GST, GST-fused bHLH, 6xHis tagged FLO2 pulled down with GST, 6xHis tagged FLO2 pulled down with GST-fused bHLH, and 6xHis-tagged FLO2 without GST-fused bHLH, respectively. Arrows indicate the size of 6xHis-tagged FLO2 (215 kD), GST-fused bHLH (48 kD), and GST (25 kD). The triangle indicates the predicted position of the 6xHis-tagged FLO2 bound with GST-fused bHLH. Size markers and their molecular sizes are shown on the left.

Figure 9.

Expression Pattern of FLO2, FLL1, and FLL2. (A) Expression of FLO2 in the stem (S), leaf blade (LB), flag leaf (FL), panicles before heading (P), 10 DAF developing seed (DS), and root (R). Amounts of transcripts are shown as the relative values to those of Actin I. Error bar shows sd (n = 3). (B) and (C) Expression level of FLL1 (B) and FLL2 (C) in the stem (S), leaf blade (LB), flag leaf (FL), panicles before heading (P), 10 DAF developing seed (DS), and root (R). Closed box, wild type (Kinmaze); open box, flo2 mutant (EM37). Amounts of transcripts are shown as the relative values to those of Actin I. Error bar shows sd (n = 3).

Figure 10.

Transgenic Lines Overexpressing FLO2. (A) Expression level of FLO2 in the developing seed at 20 DAF of the flo2 mutant (EM37), the wild type (Kinmaze), a representative CL, and three overexpression lines (OX) in which the wild-type FLO2 gene including its own promoter was introduced in the flo2 mutant. The number of each OX line is indicated. The amount of the FLO2 transcript was determined by real-time RT-PCR and shown as relative values of the FLO2 transcript to those of rice Actin I. Error bar shows sd (n = 3). (B) Shapes of the grains of the wild type and OX (line 2). The top panel indicates the pictures of grains. The bottom panel shows the grains of the wild type (Kinmaze) and the OX line from a style side. Bars = 0.1 cm. (C) to (F) Averaged grain length (C), grain width (D), grain depth (E), and grain weight (F) of these lines. Error bar shows sd (n = 20). (G) Expression levels of representative genes in the flo2 mutant, wild-type, CL, and OX lines. Total RNA extracted from 10 DAF developing seeds was used for real-time RT-PCR analysis. GluA1, glutelin A1; RA16, a 16-kD rice allergenic protein. Expression levels of the genes are shown normalized to the wild type, which is set at 1. Error bar shows sd (n = 3).

Figure 11.

Expression of FLO2, FLL1, and FLL2 during Seed Development in Normal and High-Temperature Environments. (A) and (B) Expression of FLO2 in Kinmaze (A) and Nipponbare (B) during seed development at 5, 7, 10, 15, 20, and 25 DAF. Closed box, the wild type (Kinmaze and Nipponbare, respectively) under the normal temperature condition; hatched box, the wild type treated with high-temperature stress after 5 DAF. Amounts of transcripts are shown normalized to Actin I, which is set at 1. Error bars show sd (n = 3). (C) and (D) Expression levels of FLL1 (C) and FLL2 (D) during seed development at 3, 4, 5, 7, 10, and 15 DAF. Closed box, the wild type (Kinmaze) under the normal temperature conditions; hatched box, the wild type treated with high-temperature stress after 5 DAF. Amounts of transcripts are shown normalized to Actin I, which is set at 1. Error bars show sd (n = 3).