DTH8 suppresses flowering in rice, influencing plant height and yield potential simultaneously

Abstract

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential.

Figure 1.

Phenotypes of Asominori and CSSL61. A, Photos of Asominori and CSSL61, taken when CSSL61 reached maturity. B, Photos of Asominori and CSSL61, taken when Asominori reached maturity. C, Main panicles of Asominori and CSSL61. D, Grains from the main panicles of Asominori and CSSL61. A to D, Asominori and CSSL61 were grown under NLD conditions. E, Photos of Asominori and CSSL61, taken under NSD conditions. A to E, The left is Asominori and right is CSSL61. F, Days to heading of Asominori and CSSL61 in different daylength conditions. G, The genotype of CSSL61.

Figure 2.

Map-based cloning of DTH8. A, Fine mapping of DTH8. B, Structure of DTH8. Vertical lines without labels represent single-base substitutions between Asominori and IR24. Small rectangular boxes and arrowheads represent deletions and insertions, respectively. C, DTH8 cDNA and predicted amino acid sequence. Letters with an asterisk indicate 1-bp deletion IR24, and the amino acids with gray background represent the conserved domains for DNA binding or protein-protein interaction. F.S. and STOP represent frameshift mutation and create premature stop codon, respectively. D, A phylogenic tree of HAP3 protein families in rice, Arabidopsis, yeast (ScHAP3, NP_009532), and human (HsNF-B, L06145). The unrooted tree was generated based on the amino acid sequences of the conserved domain using the program DNAMAN. The scale represents the substitution percentage per site, and the similarity to DTH8 in the conserved domain of each HAP protein is indicated in parentheses.

Figure 3.

Comparison of phenotype of the DTH8 transformants and CSSL61 plants. A, Transgene-positive T1 plant (left) and CSSL61 plant (right). B, Transgene-positive T1 plant (left) and transgene-negative T1 plant (right). C, Main panicles of transgene-positive T1 plant (left) and transgene-negative T1 plant (right).

Figure 4.

Expression pattern of DTH8 and subcellular localization of its protein. A, Tissue-specific expression pattern revealed by QRT-PCR. RNA was isolated from leaves (L), leaf sheaths (S), culms (C), young panicles (P), and roots (R) from Asominori when it was heading in NLD conditions. B, GUS expression in different tissues driven by DTH8 promoter under NLD conditions. a to f, Root, culm, node, leaf blade, leaf sheath, and young panicles. C, Nuclear localization of DTH8. a and e, Bright-field images of onion epidermal cells. b, Nuclear localization of DTH8-GFP fluorescence. c, The same cells stained with DAPI. d, The merged image of a, b, and c. f, Onion epidermal cells bombarded with the construct having GFP alone as the control. g, The merged image of e and f.

Figure 5.

The expression of DTH8 and other flowering-time genes in Asominori and CSSL61. QRT-PCR was performed with total RNA from leaves of 40-d-old plants under SD and LD conditions. Samples were collected at the initiation of the light phase (ZT 0 h). These experiments were repeated at least three times.

Figure 6.

The expression of DTH8 and other flowering-time genes in the NILs of Hd1 and Ghd7 under LD conditions. A, The expression in cv Nipponbare (Nip) and NIL (hd1; carries hd1 introgressed from cv Kasalath in a cv Nipponbare genetic background). B, The expression in NIL (Ghd7; carries functional Ghd7 from Minghui63) and NIL (ghd7; carries nonfunctional ghd7 from Zhenshan97). QRT-PCR was performed with total RNA from leaves of 40-d-old plants under LD conditions. Samples were collected at the initiation of the light phase (ZT 0 h). These experiments were repeated at least three times.

Figure 7.

The differences of the culms and scanning electron microscopic (SEM) observation of internodes between Asominori and CSSL61 plants. A, Main culms of Asominori (left) and CSSL61 (right) plants. Arrows indicate the positions of nodes. B, The differences of panicles and internodes of main culms between Asominori (left) and CSSL61 (right) plants. P, Panicle. Those from I to V indicate the corresponding internodes from top to bottom. C, Total cell number of internodes III and IV in y axis. D and E, SEM of transverse sections of the middle part of internode IV of Asominori (D) and CSSL61 (E) plants at the mature stage. F and G, SEM of longitudinal sections of the middle part of internode IV of Asominori (F) and CSSL61 (G) plants at the mature stage.

Figure 8.

Different types of DTH8 and their relationships with PS of flowering time. Type 1 is the genotype of cv Asominori. Polymorphic nucleotides in other cultivars are indicated by different colors. Deletion and insertion sites are indicated by white and black arrowheads, respectively. The number of cultivars and the mean value of PS index with each type of sequence (types from 1–9) are shown in the column at the right, with the numbers for loss-of-function types in red. The amino acid sequences of types 1 and 2 are identical. F.S. is frameshift and STOP is premature stop.

Figure 9.

A proposed model for the initiation and integration of the flowering pathways in rice under LD conditions.