Available online 10 March 2023

Author links open overlay panel, , , , , , , , , , Abstract

CONSTANS, CO-like and TOC1 (CCT) family genes play important roles in regulating heading date, which exerts a large impact on the regional and seasonal adaptation of rice. Previous studies have shown that Grain number, plant height, and heading date2 (Ghd2) exhibits a negative response to drought stress by directly upregulating Rubisco activase and exerting a negative effect on heading date. However, the target gene of Ghd2 regulating heading date is still unknown. In this study, CO3 is identified by analyzing ChIP-seq data. Ghd2 activates CO3 expression by binding to the CO3 promoter through its CCT domain. EMSA experiments show that the motif CCACTA in the CO3 promoter was recognized by Ghd2. A comparison of the heading dates among plants with CO3 knocked out or overexpressed and double mutants overexpressing Ghd2 with CO3 knocked out shows that CO3 negatively and constantly regulates flowering by repressing the transcription of Ehd1, Hd3a and RFT1. In addition, the target genes of CO3 are explored via a comprehensive analysis of DAP-seq data and RNA-seq data. Taken together, these results suggest that Ghd2 directly binds to the downstream gene CO3, and the Ghd2-CO3 module constantly delays heading date via the Ehd1 mediated pathway.

Section snippetsCRediT authorship contribution statement

Xiaowei Fan: Conceptualization, Investigation, Validation, Writing – original draft. Pengfei Wang: Formal analysis, Data curation. Feixiang Qi, Zhanyi Zhang: Investigation, Validation. Yong Hu, Shuangle Li, Jia Zhang: Conceptualization, Methodology. Liwen Liang, Juhong Liu, Lizhong Xiong: Resources. Yongzhong Xing: Conceptualization, Writing - review & editing, Supervision, Project administration, Funding acquisition.

CO3 is a potential target of Ghd2

To explore the Ghd2 target regulating the heading date in rice, ChIP-seq experiments were performed with Ghd2-flag plants. Based on ChIP-seq data from the two biological repeats, 6465 and 7831 Ghd2 binding sites were identified. The peak summits were found in introns, exons, and intergenic regions, with the greatest number of peaks found in gene promoters (Fig. 1A). The genome-wide distribution profile showed that the peaks were highly centered around the transcriptional start sites (TSS) of

The Ghd2-CO3 module constantly delays heading date through Ehd1 mediated pathway in rice

To date, many genes involved in photoperiodic flowering have been cloned in rice (Zhou et al., 2021a). Among these genes, some genes regulate only the heading date under LD or SD conditions or exhibit opposite functions under LD and SD conditions, while others play the same role regardless of the day length. A previous study suggested that Ghd2 functions as a suppressor of Ehd1 expression, thereby delaying heading under LD conditions (Shen et al., 2017). In the present study, we found that Ghd2

Vector construction and generation of transgenic rice plants

Transgenic Ghd2-OE (overexpressed Ghd2) and Ghd2-flag (overexpressed Ghd2 fused with 3×Flag) plants were generated in a previous study (Liu et al., 2016b). Knockout mutants of Ghd2 (Ghd2-CR) with a 1-bp insertion or 5-bp deletion in the coding region were also generated as described in our previous study (Zhang et al., 2021).

The cDNA sequence of CO3 (LOC_Os09g06464) was inserted into overexpression vector 1301U to generate the vector CO3-OE. The CO3-knockout vector CO3-CR was constructed using

Data availability

All raw reads generated for the materials in the study have been deposited in the National Genomics Data Center under BioProject PRJCA014274.

Uncited reference

Ecker Joseph et al., 2017, Hills, 2016.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by the Natural Science Foundation of China (U20A2031) and the Earmarked Fund for China Agriculture Research System (CARS-01). The computations in this paper were run with the bioinformatics computing platform of the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

References (60)Y. Cai et al.Dlf1, a wrky transcription factor, is involved in the control of flowering time and plant height in rice

Plos One

(2014)

J. Chai et al.Osre1 interacts with osrip1 to regulate rice heading date by finely modulating ehd1 expression

Plant biotechnology journal

(2021)

L.-H. Cho et al.Homodimerization of ehd1 is required to induce flowering in rice

Plant Physiol

(2016)

K. Doi et al.Ehd1, a b-type response regulator in rice, confers short-day promotion of flowering and controls ft-like gene expression independently of hd1

Genes & development

(2004)

M.-Y. Dong et al.Analyses of open-access multi-omics data sets reveal genetic and expression characteristics of maize zmcct family genes

AoB Plants

(2021)

R. Ecker, Joseph et al.Mapping genome-wide transcription-factor binding sites using dap-seq

Nature Protocols Erecipes for Researchers

(2017)

C. Engler et al.Golden gate shuffling: A one-pot DNA shuffling method based on type iis restriction enzymes

PLoS ONE

(2009)

C. Engler et al.A one pot, one step, precision cloning method with high throughput capability

PLoS ONE

(2008)

X. Fan et al.A long transcript mutant of the rubisco activase gene rca upregulated by the transcription factor ghd2 enhances drought tolerance in rice

The Plant Journal

(2022)

X. Gao et al.At elevated temperatures, heat shock protein genes show altered ratios of different rnas and expression of new rnas, including several novel hspb1 mrnas encoding hsp27 protein isoforms

Experimental and Therapeutic Medicine

(2021)

A. Hendelman et al.Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection

Cell

(2021)

CB,CD HillsGenetic architecture of flowering phenology in cereals and opportunities for crop improvement

FRONT PLANT SCI

(2016)

S. Jang et al.Arabidopsis cop1 shapes the temporal pattern of co accumulation conferring a photoperiodic flowering response

The EMBO journal

(2008)

S., K. Kim et al.Osco3, a constans-like gene, controls flowering by negatively regulating the expression of ft-like genes under sd conditions in rice

PLANTA -BERLIN-

(2008)

S. Laubinger et al.Arabidopsis spa proteins regulate photoperiodic flowering and interact with the floral inducer constans to regulate its stability

Development

(2006)

Y.S. Lee et al.Oscol4 is a constitutive flowering repressor upstream of ehd1 and downstream of osphyb

The Plant Journal

(2010)

S. Li et al.The amino acid residue e96 orf ghd8 is crucial for the formation of the flowering repression complex ghd7‐ghd8‐oshap5c in rice

Journal of Integrative Plant Biology

(2022)

Y. Li et al.CCT family genes in cereal crops: A current overview

The Crop Journal

(2017)

Y. Li et al.Genome-wide identification and characterization of the cct gene family in foxtail millet (setaria italica) response to diurnal rhythm and abiotic stress

Genes

(2022)

L. Liang et al.The transcriptional repressor osprr73 links circadian clock and photoperiod pathway to control heading date in rice

Plant, Cell & Environment

(2021)

View full text

© 2023 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press.