Strong photoperiod sensitivity is controlled by cooperation and competition among Hd1, Ghd7 and DTH8 in rice heading

Abstract

Rice (Oryza sativa) is a short-day (SD) plant originally having strong photoperiod sensitivity (PS), with SDs promoting and long days (LDs) suppressing flowering. Although the evolution of PS in rice has been extensively studied, there are few studies that combine the genetic effects and underlying mechanism of different PS gene combinations with variations in PS. We created a set of isogenic lines among the core PS-flowering genes Hd1, Ghd7 and DTH8 using CRISPR mutagenesis, to systematically dissect their genetic relationships under different day-lengths. We investigated their monogenic, digenic, and trigenic effects on target gene regulation and PS variation. We found that Hd1 and Ghd7 have the primary functions for promoting and repressing flowering, respectively, regardless of day-length. However, under LD conditions, Hd1 promotes Ghd7 expression and is recruited by Ghd7 and/or DTH8 to form repressive complexes that collaboratively suppress the Ehd1-Hd3a/RFT1 pathway to block heading, but under SD conditions Hd1 competes with the complexes to promote Hd3a/RFT1 expression, playing a tradeoff relationship with PS flowering. Natural allelic variations of Hd1, Ghd7 and DTH8 in rice populations have resulted in various PS performances. Our findings reveal that rice PS flowering is controlled by crosstalk of two modules - Hd1-Hd3a/RFT1 in SD conditions and (Hd1/Ghd7/DTH8)-Ehd1-Hd3a/RFT1 in LD conditions - and the divergences of these genes provide the basis for rice adaptation to broad regions.

Keywords: flowering time; heading date; photoperiod sensitivity; rice; short-day plant.

Fig. 1

Characterization of Hd1 protein. (a) Subcellular localization of Hd1‐eGFP fusion protein in rice protoplasts. The TDR‐RFP fusion protein served as the nuclear marker. Bars, 10 μm. (b) Transcriptional activation assays of Hd1 and its truncated derivatives in the yeast GAL4 system. BD is the GAL4‐DNA‐binding domain. The red Q indicates glutamine (Gln). SD‐LW, SD‐Leu‐Trp. SD‐LWHA, SD‐Leu‐Trp‐His + Ade.

Fig. 2

Eight isogenic lines with combinations of the Hd1, Ghd7 and DTH8 alleles generated by gene knockout and crossing. (a) Phenotypes of the lines (97‐d‐old plants) under artificial short‐day (ASD), natural short‐day (NSD) and artificial long‐day (ALD) conditions in Guangzhou. Bars, 25 cm. In NHLD background (HGD‐1), which has strong photoperiod sensitivity (PS), Hd1, Ghd7 and DTH8 were knocked out, respectively, via CRISPR/Cas9 to generate mutant lines (hGD‐2, HgD‐3, HGd‐4). Then a set of eight isogenic lines with all combinations of Hd1, Ghd7 and DTH8 alleles were developed by crossing among hGD‐2, HgD‐3 and HGd‐4 to generate hGd‐5, hgD‐6, Hgd‐7 and hgd‐8”. (b) Heading dates of the lines in the different day‐length conditions, and their modified PS index (mPSI) values based on ASD (a) or NSD conditions (b). Data are means ± SD (n = 50). The degrees of PS are classified into very strong (purple), strong (red), moderate (blue), and weak (green) based on the mPSI values.

Fig. 3

Heading dates and expression of Ehd1, Hd3a and RFT1 in the eight isogenic lines under artificial short‐day (ASD) and artificial long‐day (ALD) conditions. Fifty‐eight‐day‐old plants were analyzed. (a) Comparisons of the effects with and without Hd1 (Hgd‐7 vs hgd‐8), and with and without the Hd1‐Ghd7 genetic interaction (HGd‐4 vs hgd‐8 & hGd‐5 & Hgd‐7). (b) Comparisons of the effects with and without DTH8 (hgD‐6 vs hgd‐8), and with and without the Hd1‐DTH8 genetic interaction (HgD‐3 vs hgd‐8 &Hgd‐7 & hgD‐6). (c) Comparisons of the effects with and without Ghd7 (hGd‐5 vs hgd‐8), and with and without the Ghd7‐DTH8 genetic interaction (hGD‐2 vs hgd‐8 & hGd‐5 & hgD‐6). (d) Comparisons of the effects with and without the Hd1‐Ghd7‐DTH8 genetic interaction (HGD‐1 vs others). ZT, Zeitgeber time. Error bars represent SD of three biological replicates.

Fig. 4

In vitro pulldown assays for interactions between and among Hd1, Ghd7 and DTH8. (a) Hd1 interacted with Ghd7. GST‐Ghd7 is used as bait. (b) Hd1 interacted with DTH8. MBP‐DTH8 is used as bait. (c) DTH8 interacted with Ghd7. GST‐Ghd7 is used as bait. (d) Interactions among Hd1, Ghd7 and DTH8. GST‐Ghd7 was used as a bait. In Test‐1, the prey MBP‐Hd1 protein was first added to the bait GST‐Ghd7. From 1× to 3×, the intensity of the anti‐MBP hybridization band of MBP‐Hd1 gradually increased, indicating that more MBP‐Hd1 was pulled down by GST‐Ghd7. After washing, the second prey, MBP‐DTH8, was added, but the amount was constant. The intensity of the hybridization band of MBP‐DTH8 was also increased, which implies that DTH8 was more likely to bind the increased Hd1, proving the formation of a three‐protein complex of Ghd7/Hd1/DTH8. In Test‐2, the prey MBP‐DTH8 was added first to the bait GST‐Ghd7. From 1× to 3×, the anti‐MBP hybridization band of MBP‐DTH8 remained unchanged, indicating that 1× of DTH8 was enough to bind to all the bait GST‐Ghd7. After washing, the second prey MBP‐Hd1 was added with a constant amount. Its hybridization band was basically unchanged, implying that Hd1 bound to Ghd7 and DTH8 with a different domain, forming a three‐protein complex. In Test‐3, 1× of prey MBP‐DTH8 and MBP‐Hd1, or 3× of prey MBP‐DTH8 and MBP‐Hd1 were added simultaneously to the reaction. It was observed that the hybridization bands of MBP‐DTH8 and MBP‐Hd1 had similar intensities. Test‐1, Test‐2 and Test‐3 together explained that Ghd7/DTH8/Hd1 formed a three‐protein complex at a ratio of 1 : 1 : 1, instead of independent pairwise interactions. It is not the case that the amount of GST‐Ghd7 was excessive, and thus a part of it bound to MBP‐Hd1 and another part of it interacted with MBP‐DTH8.

Fig. 5

Hd1, Ghd7 and DTH8 allele combinations confer variations in rice photoperiod sensitivity (PS) and contribute to geographical adaptation. (a, b) Modified PS index (mPSI) of landraces (a) and modern indica varieties (b), which are mainly distributed in South China, and modern japonica varieties (b) in Middle and North/Northeast China. The functional types of the genes (H, G, D) include various haplotypes that may have normal‐ or reduced‐function variations. The nonfunctional alleles (h, g, d) were derived from mutations causing frame‐shift. The mPSI values in (a) were measured with heading dates under artificial long‐day (ALD) and natural short‐day (NSD) conditions, and those in (b) were based on heading dates under ALD and artificial short‐day (ASD) conditions for japonica varieties and NLD and NSD conditions for indica varieties. (c) Adaptation of rice varieties to different day‐lengths and cropping systems in the main rice‐growing areas in China. In the South China (Guangzhou is indicated with a red dot) rice‐growing area where mainly indica varieties are cultivated, those landraces (carrying the HGD‐type) with strong photoperiod sensitivity (sPS) are planted in the late season (with SD conditions), and some landraces and most modern varieties (mainly containing the hGd‐type) having weak PS (wPS) are planted in the early season (with LD conditions) and the late season. In Middle China rice‐growing areas where both indica and japonica varieties are cultivated, indica varieties having wPS are planted in the early season (with LD conditions) and the late season (with SD conditions), or in the mid‐season (with SD conditions, heading in late August to early September). And japonica varieties having sPS or moderate PS (mPS) (mainly containing the HGD‐type or hGD‐type) are planted the in the mid‐season (with SD conditions, heading in early September). In North and Northeast China rice‐growing areas, only japonica varieties having wPS are planted in the mid‐season (with LD conditions, heading in early to mid‐August).

Fig. 6

A working model of the interactions among Hd1, Ghd7 and DTH8 for regulating heading date and photoperiod sensitivity (PS) in rice. (a) In the hd1/ghd7/dth8 genotype, the basal expressions of Ehd1 and Hd3a/RFT1 are regulated by other pathways. ‘Ehd1/Hd3a/RFT1’ means that expression of these genes is regulated independently by the upstream regulator(s), or Ehd1 is first regulated and then Hd3a/RFT1 are regulated by Ehd1, or both ways. In the absence of functional Ghd7 and DTH8 alleles, Hd1 shows a primary role of promoting Hd3a/RFT1 expressions and heading under both short‐day (SD) and long‐day (LD) conditions. Ghd7 (hGd) or DTH8 (hgD) alone have weak effect on suppressing heading, mainly in LD conditions. In various combinations among the Hd1, Ghd7 and DTH8 alleles, under LD conditions Hd1 promotes Ghd7 expression and is also recruited by Ghd7 and/or DTH8 to form repressive complexes that have differently enhanced suppression effects on the Ehd1‐Hd3a/RFT1 pathway for repressing heading. In SD conditions, owing to the weakened suppression effect of Ghd7, the repressive functions of these complexes are decreased, and Hd1 competes with the complexes to promoting heading with reduced extents. The interactions of the different gene combinations (including multiple allelic variations of the genes) produce various extents of suppression and promotion effects on heading in response to different day‐lengths, and thus control different heading dates and confer various degrees of PS in rice populations. The different sizes of the marks for the proteins and promotion and repression indicate their relatively different levels of effect. Notably, the HGD working model we proposed for the PS control is based on the presence of other possibly essential factor(s) that are involved in photoperiodic flowering regulation in rice. (b) Simplified rice flowering regulatory pathways mediated commonly by Hd1 in LD and SD conditions. Other known factors related to these two pathways are omitted.