Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day-length response

Abstract

The alteration of photoperiod sensitivity has let breeders diversify flowering time in Oryza sativa (rice) and develop cultivars adjusted to a range of growing season periods. Map-based cloning revealed that the rice flowering-time quantitative trait locus (QTL) Heading date 16 (Hd16) encodes a casein kinase-I protein. One non-synonymous substitution in Hd16 resulted in decreased photoperiod sensitivity in rice, and this substitution occurred naturally in an old rice cultivar. By using near-isogenic lines with functional or deficient alleles of several rice flowering-time genes, we observed significant digenetic interactions between Hd16 and four other flowering-time genes (Ghd7, Hd1, DTH8 and Hd2). In a near-isogenic line with the weak-photoperiod-sensitivity allele of Hd16, transcription levels of Ehd1, Hd3a, and RFT1 increased under long-day conditions, and transcription levels of Hd3a and RFT1 decreased under short-day conditions. Expression analysis under continuous light and dark conditions showed that Hd16 was not likely to be associated with circadian clock regulation. Biochemical characterization indicated that the functional Hd16 recombinant protein specifically phosphorylated Ghd7. These results demonstrate that Hd16 acts as an inhibitor in the rice flowering pathway by enhancing the photoperiod response as a result of the phosphorylation of Ghd7.

Keywords: Oryza sativa L.; casein kinase I; flowering time; natural variation; photoperiod sensitivity.

Figure 1

Phenotypes and genotypes of Nipponbare, Koshihikari, a Nipponbare near-isogenic line (NIL), N-NIL(Hd16), and a Koshihikari NIL, K-NIL(Hd16). (a) Typical plants of Nipponbare, Koshihikari, N-NIL(Hd16) and K-NIL(Hd16), grown under natural day-length (ND) conditions after the flowering of Nipponbare. (b) Days to flowering under different photoperiod conditions. Values are means ± standard deviation (n = 10). SD, short-day conditions (10 h of light/14 h of dark); LD, long-day conditions (14.5 h of light/9.5 h of dark). (c) Graphical representations of the genotypes of the parent cultivars and the NILs. The 12 vertical bars represent the rice chromosomes. White and red bars indicate the Nipponbare and Koshihikari chromosome regions, respectively. Arrows indicate the positions of the Hd16 locus.

Figure 2

Map-based cloning of Hd16. (a) Chromosomal location and high-resolution linkage map of Hd16 on rice chromosome 3. The pentagons indicate candidate open reading frames within the 29.4-kbp candidate quantitative trait locus (QTL) region. The red arrowheads indicate the position of a single non-synonymous nucleotide substitution. (b) Structure of Hd16, which consists of 17 exons (gray boxes) and 16 introns. (c) Partial alignment of non-synonymous nucleotide substitutions in casein kinase isoforms. The red box indicates the tau mutation site, and the aqua box indicates the Koshihikari mutation site. The accession numbers and entire alignments are described in Figure S1. (d) Days to flowering (DTF) of three independent T₂ lines homozygous for the Hd16 transgene from Nipponbare under short-day (SD) and long-day (LD) conditions. VC, vector control. (e) Mean values and standard deviations for DTF of three independent T₂ lines for knock-down constructs of Hd16 under SD and LD conditions.

Figure 3

Effects of the Hd16 allele on DTF in backgrounds with functional and defective alleles of (a) Hd1, (b) Ghd7, (c) DTH8 and (d) Hd2. IC, isogenic control. The phenotypes of each line are presented under short-day (SD) and long-day (LD) conditions. + and − represent functional and defective alleles, respectively. N and K are the Nipponbare and Koshihikari alleles of the Hd16 gene, respectively. Values are means ± standard deviations (n = 10). Means followed by different letters are significantly different among Koshihikari and near-isogenic lines (NILs) at P < 0.05 by Tukey’s honestly significant difference test (a–d, SD conditions; e–h, LD condition). Red and underlined characters indicate defective alleles of each gene.

Figure 4

Diurnal changes in transcript levels of Hd16 and nine other genes related to rice flowering under short-day (SD) and long-day (LD) conditions in Nipponbare and N-NIL(Hd16). Transcription levels were investigated every 3 h from 14-day-old plants grown under SD conditions and from 30-day-old plants grown under LD conditions. Values are means ± standard deviations of three biological replicates. UBQ, ubiqutin used to normalize the values; ZT, Zeitgeber time.

Figure 5

Autoradiographs of the SDS-PAGE analyses of (a) phosvitin and (b) rGhd7 after in vitro rHd16 phosphorylation assays. The black triangles on the autoradiograph represent concentrations of substrates and rHd16s.

Figure 6

Yeast two-hybrid interaction between Hd16 and Ghd7. (a) Schematic diagrams of the Ghd7 fragments used in the yeast two-hybrid interaction assay. Numbers in parentheses indicate the number of amino acid residues (aa). (b) Results of the yeast two-hybrid interaction assay between Hd16 and Ghd7. Yeast strain AH109 was transformed with the indicated plasmid combinations of the bait and the prey, respectively. Values are means ± standard deviations of β-galactosidase activities from three individual colonies. pGBKT7, bait vector; pGADT7, prey vector; Hd16(Ni), Nipponbare Hd16 protein; Hd16(Ko), Koshihikari Hd16 protein.

Figure 7

Schematic representation of the proposed model of Hd16/EL1 in the part of the gene regulatory network that controls photoperiodic flowering under long-day conditions in rice. It is likely that Hd16/EL1 does not control the circadian clock. Phosphorylation of Ghd7 by Hd16/EL1 controls the transcriptional level of Ehd1. Pointed arrows indicate the upregulation of a gene; blunt-ended arrows indicate the downregulation of a gene.