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. 2024 Nov 15;17:70. doi: 10.1186/s12284-024-00750-8

OsIAA23 Promotes Heading by Directly Downregulating Ghd7 in rice

Jia Zhang 1,2,#, Wei Hu 1,3,#, Qingli Wen 1, Xiaowei Fan 1, Yong Hu 1, Qin He 1, Li Lu 1, Jinfeng Li 2, Yongzhong Xing 1,4,✉
PMCID: PMC11564490  PMID: 39542957

Abstract

Ghd7 is a central regulator to multiple growth and development processes in rice. While it is not clear how Ghd7 is regulated by upstream factors. To identify its upstream regulator, the truncated Ghd7 promoter fragments were used to screen cis elements binding to rice total nuclear proteins. Electrophoretic mobility shift assays screened one truncated fragment f3 binding to the proteins. Subsequently, the fragment f3 was employed to screen a yeast one-hybrid library, and a transcription factor OsIAA23 was screened as a direct upstream regulator of Ghd7. Dual-luciferase transient assay demonstrated the transcriptional repression effect of OsIAA23 on the activity of Ghd7, and the location of the cis elements binding to OsIAA23 in the region 1264 to 1255 bp upstream of ATG. Genetic analysis between the wild type Ghd7-OsIAA23 and single/double mutants further verified that OsIAA23 downregulated Ghd7 expression and led to a delayed heading under long day conditions. Moreover, natural variations in fragment f3 were associated with heading and geographic distribution in rice. This study sheds light on the direct regulatory mechanism of OsIAA23 on Ghd7, which enriches the understanding of the Ghd7 involved flowering regulatory network in rice.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12284-024-00750-8.

Keywords: Truncated promoter fragments, Cis element, OsIAA23, Transcriptional suppression, Upstream regulator

Introduction

Rice (Oryza sativa L.), a crucial food crop, feeds billions of people worldwide (Wu et al. 2023). It is a short-day plant, in which short day length drives its flowering. Heading date is a key agronomic trait regulated by various quantitative trait loci (QTLs), and it determines rice diversification, domestication, and grain yield (Hu et al. 2021b; Izawa 2007). Ghd7 encoding a CCT-domain containing protein was identified through a map-based cloning approach. It increases stem diameter, plant height, and grain yield by delaying heading date in the Zhenshan 97 background under long-day (LD) conditions (Xing et al. 2002; Xue et al. 2008; Yu et al. 2002). Ghd7 acts as a key regulator of multiple pathways such as photoperiod sensitivity, hormone signaling, and stress responses (Weng et al. 2014). Ghd7 also regulates seed germination by controlling the levels of abscisic acid and gibberellins, and decreases the chlorophyll content, mostly by downregulating the expression of genes involved in chlorophyll and chloroplast synthesis (Hu et al. 2021a; Wang et al. 2015). Ghd7 is critical for the adaptation of rice plants to changing environmental conditions, and it has significant implications for rice breeding (Weng et al. 2014; Zhou et al. 2021). Nonfunctional alleles of Ghd7 is associated with the adaptation of rice to cropping regions with lower temperatures and shorter growth seasons such as high-latitude China (Northeast and Northern China) and early cropping season in double rice cropping season areas (Xue et al. 2008; Zhang et al. 2015; Zong et al. 2021). Therefore, understanding the upstream regulatory mechanisms of Ghd7 is essential for developing rice varieties to enhance productivity and adaptablility to changing environmental conditions.

The regulatory network of rice heading date has been well characterized since more and more flowering genes were isolated. Rice photoperiod flowering has reported two separate pathways, OsGI-Hd1-Hd3a/RFT1 and OsGI-Ghd7-Ehd1-Hd3a/RFT1. The Arabidopsis GI-CO-FT pathway was adopted to evolutionarily conserve the OsGI-Hd1-Hd3a/RFT1 pathway, and the OsGI-Ghd7-Ehd1-Hd3a/RFT1 pathway is distinct in rice (Izawa et al. 2002; Izawa 2007). In OsGI-Ghd7-Ehd1-Hd3a/RFT1 pathway, Ghd7 greatly inhibits Ehd1 under non-inductive long-day conditions (Xue et al. 2008; Zheng et al. 2019). Currently, it has been found that some regulators control rice flowering through Ghd7, but they are not direct upstream transcription factors of Ghd7. For instance, the activation of Ghd7 is triggered by phytochrome signaling, Ehd3, Hd17, and OsCOL16, while it is inhibited by OsTrx1 (Choi et al. 2014; Itoh et al. 2010; Wu et al. 2017). The histone deacetylase HDA703 positively regulates rice brassinosteroid signaling, growth, and heading date by interacting with OsBZR1 and repressing the expression of Ghd7 (Wang et al. 2020). Ghd7 is also phosphorylated by Hd16, a casein kinase I like protein acting as a flowering inhibitor (Hori et al. 2013). Ghd7 was considered to be a bridge linked Hd1- and Ehd1-mediated flowering pathways, since Ghd7 interacts with Hd1 and particularly specifically binds to a cis-regulatory area in Ehd1 and restricts its expression (Nemoto et al. 2016; Zhang et al. 2017). Ghd7 also has a greatly diverse promoter, and the functional C/T mutation in the promoter area is connected with plant height possibly through changing gene expression (Lu et al. 2012).

The IAA protein is widely present in plants as a transcriptional repressor in the signal transduction of plant growth hormones, and plays an important role in the early response to auxin (Guilfoyle and Hagen 2007; Hagen and Guilfoyle 2002). The IAA protein has specific structural domains that interact with ARF proteins (auxin response factors) to regulate downstream genes (Liscum and Reed 2002). IAA genes have diverse roles in multiple developmental processes such as embryonic development, lateral root initiation and elongation, hypocotyl growth, directional movement, and flower organ development (Luo et al. 2018; Lau et al. 2008). The rice IAA gene family has 31 members. OsIAA1 participates in the response to auxin and brassinosteroid hormones, and exert a significant effect on rice light response and embryonic sheath elongation (Song et al. 2009; Thakur et al. 2001). Overexpression of OsIAA4 results in dwarfism, increased tiller angle, and weakened gravity response (Song and Xu 2013). OsIAA3, OsIAA11, and OsIAA13 are involved in rice lateral root development. OsIAA3 also regulates rice grain length through interaction with OsARF25 and Gnp4/LAX2 (Kitomi et al. 2012; Zhang et al. 2018; Zhu et al. 2012). The OsIAA6 protein exerts an effect on both drought stress response and tiller development control (Jung et al. 2015). The P2 protein of rice dwarf virus (RDV) is easier to invade and replicate in rice plants after interacting with OsIAA10 (Jin et al. 2016). OsIAA23 affects rice root and shoot development, plant height, reproductive capacity, and maintenance of the quiescent center (QC) of the root tip (Jiang et al. 2019). Currently, the functions of most IAA family genes in rice are not clear, and no studies report IAA family genes regulating rice flowering.

Yeast one-hybrid (Y1H) assay successfully screens trans regulators of target genes from a cDNA library. However, this technology frequently uses a long promoter fragment to screen too many transcription factors, which can impede further confirmation and validation (Ben Daniel et al. 2016). In this study, we employed truncated short fragments of the Ghd7 promoter for Y1H assay to screen the potential trans regulator of Ghd7 ( LOC_Os07g15770). The fragment f3 fished a protein OsIAA23 (LOC_Os06g39590), which was confirmed by both EMSA and genetic analysis. OsIAA23 binds to the promoter of Ghd7 and inhibits the transcriptional activity, which ultimately represses flowering in rice.

Results

Possible cis Elements in the Promoter of Ghd7

Promoter prediction in the PLACE platform (https://www.dna.affrc.go.jp/PLACE/?action=newplace) revealed that the Ghd7 promoter contains 270 cis elements, posing a challenge to identify certain transcription factors binding to a specific Ghd7 promoter fragment (Table S1). To improve the efficiency and accuracy of assessing the potential transcription factors binding to Ghd7 by Y1H assays, the truncated Ghd7 promoter fragments were used to test their binding activity to proteins. Firstly, 1.5 kb promoter of Ghd7 was divided into 8 fragments (P1–P8) each with about 200 bp, then the fragments were synthesized and labeled with 5’ biotin for electrophoretic mobility shift assay (EMSA) with rice total nuclear proteins. Three fragments (P-1, P-3, and P-7) showing protein binding activity were further divided into fragments of about 50 bp each (Fig. 1A). Short DNA fragments approximate 50 bases in length were amplified with biotin labeled primers for EMSA. Finally, only one fragment, f3 (50 bp from 1275 to 1226 bp upstream to ATG), interacted with rice total nuclear proteins, indicating that f3 contains potential cis elements for binding upstream transcription factor (Fig. 1B).

Fig. 1.

Fig. 1

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Screening of potential transcription factor binding sites on Ghd7 promoter. (A) Distribution of Ghd7 promoter fragments used in EMSA. (B) EMSA was carried by using the rice total nuclear protein and the f3 with 5’ Bio. Competition for the labeled sequences was tested by adding different concentrations of unlabeled probe. (C) Schematic map of CRISPR/Cas9-induced f3 region of the Ghd7 promoter mutants. The sgRNA target site is shown in blue, and the mutation site are shown in red, where “-” represents deletion mutation. (D) Days to heading of the wild-type and f3 mutant plants under LD conditions, **P < 0.01. (E) Ghd7 expression level in the f3 mutant plants under LD conditions, **P < 0.01

A total of 16 potential cis elements were predicted by PLACE in seven regions of fragment f3. Most of them are related to light regulation such as SORLIP1AT, GT1CONSENSUS, and IBOXCORE (Table S2 and Fig. S1). To investigate whether f3 is associated with rice flowering, a CRISPR/Cas9 construct was designed to generate mutations in the f3 region. Through Agrobacterium-mediated transformation, 3 mutants were generated from independent transgenic plants, with deletion of 4, 31 and 107 bp (Fig. 1C). The smaller segments happen to be encompassed within the larger segments. All these mutant (T2) lines headed approximately two days later than the wild type under LD conditions (Fig. 1D). Besides, the upregulation of Ghd7 was observed in the f3 mutants when compared to the wild type under LD conditions, which conformed to later heading of mutant plants (Fig. 1E).

Potential Transcription Factors Binding to the Promoter of Ghd7

The fragment f3 was utilized as a bait to screen the upstream transcription factor of Ghd7 from a Y1H library. OsIAA23 was identified and further verified on selective medium (Fig. 2A). The co-transformants of f3-AbAi along with pGADT7-Rec-OsIAA23 could grow on the synthetic dextrose SD/-Leu medium with 200 ng/mL 50 mM AbA, which was like the positive control (with p53-AbAi and pGADT7-Rec-p53). While the negative control (with f3-AbAi and pGADT7-Rec-p53) could not grow. Then the full-length coding sequence of OsIAA23 was cloned and expressed in E. coli. The expressed OsIAA23 protein was purified and confirmed by SDS-PAGE analysis (Fig. 2B). Then, the purified OsIAA23 protein and the biotin-labeled f3 fragment were used to perform EMSA, which showed the specific bound of OsIAA23 protein to the f3 fragment (Fig. 2C). Thus, OsIAA23 may serve as the upstream transcription factor for Ghd7.

Fig. 2.

Fig. 2

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OsIAA23 interacts with f3 in vitro. (A) OsIAA23 bound to the f3 in yeast cells through a yeast one-hybrid assay. (B) Protein prokaryotic expression of OsIAA23. (C) EMSA was carried by the OsIAA23 protein and f3 labeled with 5’ Biotin. Competition for the labeled sequences was tested by adding different concentrations of unlabeled probes

OsIAA23 Acting as a Transcriptional Repressor of Ghd7

To determine the subcellular localization of OsIAA23, the complete protein sequence of OsIAA23 was linked to the YFP reporter gene under the control of the CaMV 35 S promoter. OsIAA23-YFP and the nuclear marker Ghd7-RFP were co-transfected into rice protoplasts, and the merged YFP fluorescence with RFP fluorescence clearly demonstrated that OsIAA23 is a nuclear protein (Fig. 3A). To discover how OsIAA23 regulates the Ghd7 transcription, OsIAA23 was used as effector, and the full promoter of Ghd7 as well as the mutated fragments with deletion of 4, 31, and 107 bp from the f3 mutants were fused to the firefly luciferase as reporters, respectively (Fig. 3B). All systems containing OsIAA23 exhibited lower relative activity of LUC compared to the control, indicates that OsIAA23 has a significant transcriptional repression effect on the activity of Ghd7 in vivo (Fig. 3C). To identify the core cis element responsible for binding OsIAA23, the activity of the full promoter was compared with that of the truncated ones. No significant change was detected in inhibitory activity in the Ghd7 promoter with the 4-bp deletion. However, the inhibition of Ghd7 by OsIAA23 was significantly reduced when the Ghd7 promoter missed 31–107 bp, indicating that OsIAA23 binds to 1261–1231 bp upstream to ATG (Fig. 3D).

Fig. 3.

Fig. 3

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Effect of OsIAA23 on promoters of Ghd7 using a dual-luciferase transient assay in rice protoplasts. (A) Subcellular localization of OsIAA23 in nucleus of rice protoplasts and Ghd7-RFP was used as a nuclear marker. (B) The main components of the vectors. (C) The relative LUC activity and (D) the LUC activation level of OsIAA23 on the promoters is compared with the control. Error bars indicate the SE of three replicates. **P < 0.01. (E) The molecular docking simulation of OsIAA23 and f3

To deeply investigate the core motif in the f3 region, we devised truncated fragments M1-M5 for EMSA experiments. The results affirmed that M1, M3, M4, and M5 exhibited specific binding to the OsIAA23 protein like the f3 fragment, whereas M2 lost its capacity binding to OsIAA23 (Fig. S2). Therefore, the core cis element specifically bound by OsIAA23 is suggested in the region missing in M2, 1264–1255 bp upstream of the ATG. Furthermore, based on the predicted 3D structure of OsIAA23 and f3 using ALPHAFOLD (https://cosmic-cryoem.org/tools/alphafold/) and Build Biopolymer from sequence, respectively, a molecular docking study was performed to deeply understand the mechanism of OsIAA23 binding to f3. The optimal conformation of the complex of OsIAA23 and f3 is depicted in Fig. 3E, and it has the highest negative free energy value of − 1231.62 kcal/mol indicates a strong binding stability between OsIAA23 and f3. Nucleotide sites in the f3 DG1(+), DC4(+), DG8(+), DG45(-), DC46(-), DA10(+), DG11(+), DG15(+), DC19(+), DA20(+), DG33(-), DC34(-), DA37(-), DG38(-), as well as amino acid sites in OsIAA23 HIS115, ALA119, LEU122, CYS121, PRO94, TRP65, LYS98, LYS87, LYS98, ARG73, ARG73, SER32, ARG44, ARG42, THR37, LYS43, ARG69 specifically interact by hydrogen bonding or salt bridge (Table S3). Combined with the result of the EMSA and dual-luciferase transient assay, the core cis element specifically bound by OsIAA23 is suggested in the region 1264–1255 bp upstream of the ATG (highlighted by the red box in Fig. 3E). In this region, the predicted specific interactions include: DA10(+) with ARG73 (2 H-bonds, 1 salt bridge), DG11(+) with ARG73 (1 salt bridge), DG15(+) with SER32 (1 H-bond), DC19(+) with ARG44 (1 H-bond), DA20(+) with ARG42 (2 H-bonds), DG33(-) with THR37 (1 H-bond), DC34(-) with LYS43 (1 H-bond), DA37(-) with ARG69 (1 H-bond, 1 salt bridge), and DG38(-) with ARG69 (1 H-bond, 1 salt bridge).

OsIAA23 Promoting Heading Date

To investigate the expression pattern of OsIAA23, we collected leaves from ZH11 plants for RNA extraction at 4-h intervals for 24 h and examined transcription level through qRT-PCR. The expression pattern of OsIAA23 is similar to that of Ghd7, reaching a peak in the morning (at 8:30) and then declining. However, the decline in Ghd7 expression nearly ceasing before dawn is significantly faster than that in OsIAA23 expression (Fig. S3). To further clarify the interaction effects between OsIAA23 and Ghd7 on heading date, the single mutants of osiaa23 and ghd7 was generated through CRISPR strategy, and the osiaa23-ghd7 double mutant was obtained by crossing the two single mutants (Fig. S4). The osiaa23 mutant (70.4 ± 1.5 d) exhibited a significant delay in flowering time in comparison to the wild type (65.6 ± 1.0 d) under LD conditions, whereas the ghd7 mutant (55.0 ± 1.9 d) displayed a remarkable promotion in flowering time. Interestingly, the osiaa23 ghd7 double mutant (54.5 ± 1.0 d) showed a similar phenotype to the ghd7 mutant, indicating that OsIAA23 function upstream of Ghd7 in regulating heading date (Fig. 4A and C). The expression levels of Ghd7 were significantly upregulated in the osiaa23 mutant compared with the wild type, while the transcript levels of Ehd1, RFT1 and Hd3a were reduced (Fig. 4D and E and Fig. S5). These findings further confirm that OsIAA23 acts as an upstream regulator of Ghd7 in the photoperiod flowering pathway by downregulating Ghd7 under LD conditions.

Fig. 4.

Fig. 4

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The phenotypes of CRISPR/Cas9 induced osiaa23 mutants. (A–C) Phenotypes of the osiaa23, ghd7 and ghd7/osiaa23 mutants under LD conditions. (D–E) Diurnal expression patterns of Ghd7 and Ehd1 in osiaa23 mutants and Zhonghua 11 under LD conditions. The black and white bars represent the dark and light periods, respectively

Linkage Disequilibrium between OsIAA23 and Fragment f3

To investigate the natural variations of OsIAA23 at protein level, a total of 528 rice accessions were included (Table S4). Five haplotypes of OsIAA23 were identified excluding rare haplotypes (less than three accessions) based on deduced amino acid sequences. Hap1 and Hap4 were the predominant haplotypes in indica rice, while Hap2 and Hap3 were the predominant haplotypes in japonica rice. Specifically, Hap5 was detected in intermediate cultivars with an average heading date of 105.5 ± 10.5 days, which significantly delayed flowering as compared with other haplotypes with an average heading date around of 95.5 days (Fig. 5A and B). This suggests that Hap5 may be a weak allele of OsIAA23.

Fig. 5.

Fig. 5

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Natural variation in OsIAA23. (A) Haplotypes of OsIAA23 in 528 accessions. (B) The heading date of OsIAA23 with different haplotypes. Data marked with different lowercase letters are significantly different, P < 0.05. (C) Geographical distribution of different OsIAA23 haplotypes. (D) Evolutionary relationships among distinct OsIAA23 haplotypes. The subgroups indica, japonica, aus, and intermediate are represented by yellow, blue, purple and pink, respectively

To better evaluate the distribution of these haplotypes in East Asia, especially in China, the Chinese germplasm resources were classified based on China’s planting regions (https://www.ricedata.cn/) and geographical divisions, including Southwest China, South China, Central China, North China, East China, Northwest China, and Northeast China. Based on the haplotype distribution table across different regions in China (Table S4), we found that Hap1 and Hap2 are the most prevalent in most regions, which is consistent with germplasm distribution trends in whole germplasm resources. Hap4 is less common in China, with only nine accessions, and Hap5 is completely absent. In the high-latitude regions of North China and Northeast China, the proportions of Hap2 and Hap3 are relatively higher; however, due to the smaller number of resources in these regions, it is difficult to determine whether Hap2 and Hap3 have been subject to selection (Fig. 5C). Besides, combining the haplotype distribution results from germplasm resources of other countries, OsIAA23 does not appear to have undergone artificial selection during breeding. Phylogenic tree analysis revealed that Hap4 is in the central position, all other haplotypes differed from Hap4 at one amino acid caused by a single base mutation (Fig. 5D). A significant linkage disequilibrium (r2 = 0.44) was detected among OsIAA23 and f3 variation sites, providing additional evidence for the strong functional correlation between these two genes (Fig. S6).

Discussion

The OsIAA family members act as transcription factors. They regulate the expression of auxin response genes to control multiple developmental processes. But nowadays, no studies reported their roles in regulating heading date. Ghd7 is one of the most crucial genes involved in regulating flowering in rice. It also plays a central role in governing the overall growth and development of rice as a pleiotropic gene (Weng et al. 2014). In this study, OsIAA23 was discovered as a direct upstream regulator of Ghd7, which inhibits the expression of Ghd7 by binding to the f3 region in the Ghd7 promoter and ultimately promotes flowering (Fig. 6). Recently, it has been demonstrated that the application of cytokinin to rice delays the heading date (Cho et al. 2022). While this study is the first time to establish a molecular foundation for the connection between auxin response and rice flowering, thereby enhances our understanding of the signaling pathway involved in heading in rice.

Fig. 6.

Fig. 6

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The Schematic model of the modular OsIAA23-Ghd7 regulating photoperiod flowering

The rice Aux/IAA gene family consists of 31 members. This suggests that OsIAA23 may be functionally redundant with other IAA proteins in Zhonghua 11 background. It is important to investigate whether other IAA proteins are involved in the regulation of flowering. Moreover, it would be beneficial to uncover whether auxins or other hormones regulate flowering, and whether some AFB or ARF proteins are responsible for the regulation of Ghd7 by OsIAA23. Furthermore, since the absence of the full f3 region did not entirely eliminate the inhibition of Ghd7 by OsIAA23, there may be additional interaction sites that allow OsIAA23 to exert partial inhibition even when the f3 fragment of the Ghd7 promoter is missing. Therefore, the exact cis element responsible for OsIAA23 binding needs to be thoroughly examined.

Recent study found that specific cis regulatory regions can be associated with different pleiotropic functions, and mutations in these regions may have less harmful effects on pleiotropy compared to mutations in protein-coding regions (Hendelman et al. 2021). This highlights the importance of studying cis regulatory regions in understanding the genetic basis of complex traits. Certainly, there should have some other direct upstream regulators of Ghd7. Besides the proposed EMSA strategy with several truncated Ghd7 promoter fragments screening rice total nucleoproteins, more special cis elements would be defined by saturated mutations in the promoter of Ghd7, in which some mutants will exhibit changing phenotype in different traits like heading date, plant height and yield. Thus, favorable mutations would be generated for rice production beside the identification of cis elements. It is expected that a more complex regulatory network involved by Ghd7 would be established with the identification of more upstream regulators.

Materials and Methods

Electrophoretic Mobility Shift Assay

To extract nuclear protein, we first ground leaves from 4-leaf-old rice plants into a fine powder using liquid nitrogen. Next, we extracted the protein using buffers 1, 2, and 3, following the same nuclear protein extraction method as in the ChIP assay (Bowler et al., 2004). The complete cDNA sequence of OsIAA23 was incorporated into the pGEX-4T-1 expression vector (GE Healthcare) using Daniel Gibson’s enzymatic assembly technique and expressed in Escherichia coli (DE3 cells from GE Healthcare) to refine the OsIAA23 protein. Glutathione HiCap Matrix (Qiagen;139302891) was was utilized for the purification of the expressed protein, and 5’Biotin oligonucleotides were synthesized by Shanghai Sangon (Table S5). The double-stranded oligonucleotides were achieved by combining equal amounts of complementary single-stranded oligonucleotides and incubating them at 95°C for 2 minutes, followed by cooling to 25°C. The LightShift Chemiluminescent EMSA Kit (Thermo Scientific) was used for EMSA analysis. A 20-minute preincubation of nuclear protein or purified GST-fused OsIAA23 was performed with the binding buffer (10 mM Tris-HCl with pH 7.5, 2.5% glycerol, 1 mM DTT, 1 ug of poly(dI-dC), 5 mM MgCl2, and 50 mM KCl) at room temperature. Next, the non-labeled probe was added to the mixture for the competition assay. After 20 minutes at room temperature, 1 uL of 5’ biotin-labeled probe was added and incubated for another 20 min at room temperature. The samples were electrophoresed on a 6% PAGE gel with 0.5×TBE buffer for 1 h at 4 °C. Electrophoretic transfer of binding reactions to nylon membrane than used UV-light to cross-link. The signal of biotin-labeled DNA was captured by Chemiluminescence.

DNA Extraction and DNA Sequencing

The cetyl-trimethyl ammonium bromide (CTAB) approach (Murray and Thompson, 1980) was employed for DNA extraction from leaf blades during the tillering phase. For DNA sequencing, we used 5 U of ExoI (NEB, Ipswich, MA, USA) and 0.13 U of shrimp alkaline phosphatase to digest an 8 µL aliquot of the PCR product. The digested product was then purified to perform the sequencing reaction using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The contigs were assembled using the SEQUENCHER 4.1.2 program, and singletons and ambiguous sites were subsequently re-sequenced. (Table S5).

Yeast One-Hybrid Assay

We used the Matchmaker Gold Yeast One-Hybrid Library Screening System (Clontech). The 50 bp target region of Ghd7 promoter was cloned into the pAbAi vector used as bait. Prey proteins, are expressed as GAL4 AD fusion protein expressed from the pGADT7-Rec vector which contains library cDNAs. The cDNA library was made from rice variety Zhonghua 11 (ZH11) leaves. The co-transformation of pGhd7-50-pAbAi and prey into the yeast strain Y1HGold (Clontech) plated on SD/-Leu /+AbA (200 ng/mL) was made to choose for colonies whose AbAr reporter has been activated by prey proteins.

Dual Luciferase Transcriptional Activity Assay in Rice Protoplasts

Rice protoplasts were separated and changed based on the polyethylene glycol-mediated approach (Xie and Yang 2013). The protoplasts were isolated from 14-d-old seedlings of ZH11 in a digestion solution containing 0.75% Macerozume R10, 0.3% Cellulase RS, 0.6 M Mannitol, 5 mM b-mercaptoethanol, 1 mM CaCl2, 0.1% BSA, and 10 mM MES (pH5.7). The isolation process took place for 5 h under dark conditions. The protoplasts were incubated in W5 solution (125 mM CaCl2, 5 mM KCl, 154 mM NaCl, and 2 mM MES with pH 5.7) for 30 min. After incubation, they were filtered through a sieve mesh and then resuspended in a solution containing 15 mM MgCl2, 0.6 mannitol, and 4 mM MES after 5-minute centrifugal at 100 g. For transformation, 3 µg of various plasmids were combined with 100 μL of protoplasts and 110 μL of a polyethylene glycol-CaCl2 solution (40% polyethylene glycol 4000, 100 mM CaCl2, and 0.6 M mannitol). After incubating at room temperature for 10 min, the process was stopped by adding 440 μL of W5 solution. Protoplasts were cultured in 800 μL of 4 mM KCl, 0.6 mannitol, and 4mM MES with pH 5.7 after being collected at 100 g for 5 min on 24-well culture plates. After 12 h, the protoplasts were collected for the dual-luciferase activity assay following the manual instructions (Promega; Dual-Luciferase Reporter Assay System).

Molecular Modeling and Docking

Online kits ALPHAFOLD (https://cosmic-cryoem.org/tools/alphafold/) was adopted to predict three-dimensional (3D) crystal structure of OsIAA23 protein. Then, the crystal structure subjected to protein preprocess, regenee states of native ligand, H-bond assignment improvement, protein energy, minimization and eliminate waters by applying Protein Preparation Wizard module in Schrodinger Suite 2023. Besides, Build Biopolymer from Sequence was used to predict the 3D structure of f3 DNA, while the Nucleotide Preparation Wizard module was used to process the tertiary structure. Next, molecular docking between OsIAA23 and f3 was performed using Schrodinger Maestro13.5 (accessed in March 2023). It was set that there are 7000 and 30 ligand rotations to probe and maximum poses to return. In additional, Protein Interaction Analysis module was utilized to identify the specific region of OsIAA23 that interacts with the f3.

Plant Materials and Growth Conditions

The vector pCXUN-Cas9 was used to generate the Ghd7 promoter mutant, and the target sequence was provided by the website http://cbi.hzau.edu.cn/cgi-bin/CRISPR (Lei et al. 2014). The target sequence started with an “A” base, as the OsU3 promoter was utilized. Therefore, gRNA expression cassettes were acquired with the overlapping PCR approach (Sun et al. 2016). The pOsU3-gRNA plasmid was used as a template for two rounds of PCR (Table S5). The gRNA fragment was then inserted into the pCXUN-Cas9 vector using Daniel Gibson’s enzymatic assembly method, which was linearized using FastDigest KpnI (Thermo Scientific, USA) (Gibson et al. 2009). The japonica rice variety ZH11(O. sativa spp. japonica) was selected as the recipient for transformation. The homozygous T2 mutants were sown on 17 May, 2017, at Huazhong Agricultural University, Wuhan, China (31°N latitude). Seven plants from each variety were planted in a single row with a spacing of 16.5 cm between plants and 26.5 cm between rows (varieties). The measurement of the heading date was made with 5 middle plants. The heading date was recorded to the number of days from sowing to the emergence of the first panicle in the plant. The phenotype data for a particular variety was determined by averaging the phenotypic values of five plants.

Haplotype Analysis

The variation of OsIAA23 and the f3 fragment of Ghd7 were obtained from the Rice Variation Map v2.0 (http://ricevarmap.ncpgr.cn/) (Zhao et al. 2015). The haplotype analysis was conducted by geneHapR package (Zhang et al. 2023), and the LD visualization was conducted by LDheatmap package (Shin et al. 2006).

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (30.3KB, xlsx)
Supplementary Material 2 (9.5KB, xlsx)
Supplementary Material 3 (12.6KB, xlsx)
Supplementary Material 4 (52.8KB, xlsx)
Supplementary Material 5 (11.2KB, xlsx)
Supplementary Material 6 (8.2MB, docx)

Acknowledgements

The authors would like to thank Mr. J.B. Wang for his hard work in the field.

Author Contributions

Y.X. conceived and designed the experiments. JZ and XF performed most the experiments. J.Z., W.H., Q.W., X.F., Y.H., Q.H., L.L., and J.L. analyzed the data. Y.X., J.Z. and W.H. wrote the paper. All authors reviewed and approved the final manuscript.

Funding

This study was supported by the Natural Science Foundation of China (32101750, U20A2031), High Quality Development Projects of Hubei Province for Seed Industry (HBZY2023B001-03) and the Earmarked Fund for China Agriculture Research System (CARS-01).

Data Availability

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jia Zhang and Wei Hu contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (30.3KB, xlsx)
Supplementary Material 2 (9.5KB, xlsx)
Supplementary Material 3 (12.6KB, xlsx)
Supplementary Material 4 (52.8KB, xlsx)
Supplementary Material 5 (11.2KB, xlsx)
Supplementary Material 6 (8.2MB, docx)

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

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