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Communication

OsBBX2 Delays Flowering by Repressing Hd3a Expression Under Long-Day Conditions in Rice

1
College of Advanced Agriculture and Ecological Environment, Heilongjiang University, Harbin 150080, China
2
State Key Laboratory of Black Soils Conservation and Utilization, Key Laboratory of Soybean Molecular Design Breeding, NortheastInstitute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China
3
University of Chinese Academy of Sciences, Beijing 100049, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(1), 48; https://doi.org/10.3390/plants14010048
Submission received: 23 November 2024 / Revised: 17 December 2024 / Accepted: 24 December 2024 / Published: 27 December 2024
(This article belongs to the Special Issue Crop Functional Genomics and Biological Breeding)

Abstract

:
Members of the B-Box (BBX) family of proteins play crucial roles in the growth and development of rice. Here, we identified a rice BBX protein, Oryza sativa BBX2 (OsBBX2), which exhibits the highest expression in the root. The transcription of OsBBX2 follows a diurnal rhythm under photoperiodic conditions, peaking at dawn. Functional analysis revealed that OsBBX2 possesses transcriptional repression activity. The BBX2 was overexpressed in the rice japonica cultivar Longjing 11 (LJ11), in which Ghd7 and PRR37 were non-functional or exhibited weak functionality. The overexpression of OsBBX2 (OsBBX2 OE) resulted in a delayed heading date under a long-day (LD) condition, whereas the bbx2 mutant exhibited flowering patterns similar to the wild type (WT). Additionally, transcripts of Ehd1, Hd3a, and RFT1 were downregulated in the OsBBX2 OE lines under the LD condition. OsBBX2 interacted with Hd1 (BBX18), and the bbx2 hd1 double mutant displayed a late flowering phenotype comparable to that of hd1. Furthermore, OsBBX2 enhanced the transcriptional repression of Hd3a through its interaction with Hd1, as demonstrated in the protoplast-based assay. Taken together, these findings suggest that the OsBBX2 delays flowering by interacting with Hd1 and co-repressing Hd3a transcription.
Keywords:
rice; heading date; BBX2; Hd1; Hd3a

The BBX family of proteins is highly conserved across green plants. They are characterized by one or two B-Box motifs in the N-terminal domain and a specific CCT (CONSTANS, CO-like, and TO) domain at the C-terminus [1]. They are involved in many plant regulatory networks, ranging from seedling photomorphogenesis to stress response [2]. In Arabidopsis thaliana, a total of 32 genes encode BBX proteins, while in rice, the BBX family comprises 30 proteins, designated as OsBBX1 to OsBBX30, based on their chromosomal locations. Among these, seven OsBBXs contain two B-box domains and a conserved CCT domain, ten possess one B-box and one CCT domain, three have a single B-box domain, and ten contain two B-box domains [1].
Most BBX family members have been reported to play a vital role in floral transition. CONSTANS (CO)/BBX1 was the first BBX family member identified in Arabidopsis. CO induces flowering by activating the expression of FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS1 (SOC1) [3]. Subsequent studies have identified several BBX proteins that regulate flowering. For instance, BBX7 (COL9) delays flowering under long-day (LD) conditions by reducing CO expression [4], while bbx4 (col3) plants flower earlier during both short and long days, and COL3 is a positive regulator of photomorphogenesis [5]. The late flowering phenotypes of overexpressed BBX32 (EIP6) transgenic plants suggest that its interaction with EMBRYONIC FLOWER1 (EMF1) can regulate flowering time in Arabidopsis [6]. BBX19 functions as a negative regulator of flowering time, and it also functions as a regulator of circadian rhythm by complexing PRR proteins to enhance their repressive effect on CCA1 transcription [7]. BBX24 and BBX6 (COL5) function as positive regulators of flowering, with BBX24 overexpressing lines exhibiting accelerated flowering by striving for FLOWERING LOCUS C (FLC) to impact downstream flowering genes [8]. BBX6 overexpression induces early flowering by promoting FT expression [9].
Rice HEADING DATE 1 (Hd1/OsBBX18), which is an orthologue of CO in Arabidopsis, promotes heading under short-day (SD) conditions and inhibiting under long-day (LD) conditions [10]. In addition, a group of B-box-containing proteins were proven to repress flowering in rice. BBX27 (OsCO3), which has two incomplete B-boxes, serves as a flowering repressor upstream of Hd3a under SD [11]. The overexpression of OsBBX26 (OsCOL15) suppresses flowering by promoting Ghd7 and repressing RID1 under both SD and LD conditions [12]. The overexpression of OsBBX17 (OsCOL16) delays heading under both SD and LD conditions by upregulating Ghd7 transcripts [13]. OsCOL13 functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1. On the other hand, OsCOL13 is functionally redundant with BBX5 (OsCOL4), which is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB [14]. Both the overexpression of BBX7 (OsCOL9) and BBX10 (OsCOL10) delay flowering under SD and LD conditions by repressing Ehd1, Hd3a, and RFT1 expression. OsCOL10 acts as a suppressor of rice flowering time by bridging Ghd7 and Ehd1 [15]. OsBBX14 suppresses rice flowering by regulating either Hd1 or Ehd1 under LD or SD conditions [16].
Here, we show that BBX2 acts as a negative regulator of flowering time under long-day (LD) conditions. BBX2 physically interacts with Hd1 to repress the transcription of Hd3a.
The BBX2 (LOC_Os02g07930) gene was overexpressed in the rice japonica cultivar Longjing 11 (LJ11), resulting in two independent BBX2 overexpressing (OE) transgenic lines, which exhibited significantly higher levels of BBX2 expression (Supplemental Figure S1A). The BBX2 OE plants flowered outstandingly later than LJ11 under the LD (14 h light/10 h dark) condition, with delays of 9.35 and 10.65 days for the two approaches (Figure 1A,B). An RT-qPCR interpretation exhibited that the mRNA levels of Ehd1, Hd3a, and RFT1 were dramatically lessened in the BBX2 OE plants compared to LJ11 (Supplemental Figure S1B–D). The decreased expression of the florigen genes and their catalyst Ehd1 is consistent with the later flowering phenotypes observed in BBX2 OE plants (Figure 1A,B).
Most BBX proteins serve as flowering repressors [2]. To inspect the attributes of BBX2, we first assessed its transcriptional activity in a rice protoplast system; BBX2 reduced LUC transcription activity compared with GAL4BD, revealing it to be a transcriptional repressor (Figure 1C,D). As BBX2 functions as a negative regulator of flowering, we investigated the diurnal expression modes of BBX2 under LD and SD conditions. The BBX2 manifestation began to accumulate slowly at dusk before peaking at dawn and sharply decreasing until dusk to its lowest level under both SD and LD conditions (Supplemental Figure S2A,B). In addition, we examined BBX2’s pattern of temporal and spatial expression in diverse tissues, including roots, stems, leaves, panicles, and seeds, by RT-qPCR. BBX2 was shown mainly in the roots, with a comparative expression in the leaves, stem, and spike, and no expression was observed in the panicles or seeds (Supplemental Figure S2C).
To analyze the underlying mechanism for BBX2 in regulating flowering, we investigated the potential interacting proteins of BBX2 by the STRING database (https://cn.string-db.org/). We first used a yeast two-hybrid system to identify whether BBX2 could physically interact with Hd1, PRR1, and DTH2. The results demonstrated that BBX2 interacts with Hd1, but not with PRR1 or DTH2 (Figure 1E). To confirm this interaction, we applied split luciferase complementation assays by creating merger constructs of BBX2 and Hd1 to C- and N-terminal fragments of luciferase. Agroinfiltration-based transient assays in N. benthamiana showed the restoration of luciferase activity in the leaves co-infiltrated with BBX2 and Hd1 (Figure 1F).
To explore the genetic relationship between BBX2 and Hd1, we generated bbx2, hd1, and bbx2 hd1 mutants using CRISPR/Cas9-mediated genome editing in the LJ11 background [17]. Target sites were designed for the first exons of BBX2 and Hd1 (Supplemental Figure S3A,B). Despite the different mutation types in the single and double mutants, BBX2 inserted a G base in the single mutant of bbx2, and a G base was missing in the bbx2 hd1 double mutant. Hd1 inserted a T base in the single mutant, and the A base was inserted in the bbx2 hd1 double mutant, for which successful gene knockout was achieved (Supplemental Figure S3C,D). Phenotypic analysis showed that bbx2 flowers at a similar time to LJ11 under the LD condition, while hd1 and bbx2 hd1 mutants flower approximately 5.1 to 5.6 days later than LJ11 under the LD condition (Figure 1G–J). These results suggest that Hd1 functions genetically downstream of BBX2. As a flowering repressor, Hd1 controls the expression of florigen genes under the LD condition. We hypothesized that BBX2 enhances the inhibitory effect on flowering by physically interacting with Hd1. Considering that BBX2 can interact with Hd1, which represses Hd3a expression, we performed a transient expression assay in rice protoplasts using 35SPro:GFP as a control and 35SPro:BBX2, 35SPro:Hd1, and Hd3aPro:LUC as effectors and reporters, respectively (Figure 1K). The results indicate that 35SPro:Hd1 efficiently suppressed Hd3a expression compared to the control 35SPro:GFP, and this suppression was further enhanced by the addition of 35SPro:BBX2 (Figure 1L).
Ehd1 and Hd1 are key factors in regulating flowering in rice, particularly under long-day conditions [18]. It has been shown that Hd1 and Ghd7 interact to form a complex, which specifically binds to the cis-regulatory region of Ehd1, thereby suppressing its expression [19]. Additionally, studies have shown interactions between Hd1, Ghd7, and PRR37. Notably, Hd1 promotes flowering in both long- and short-day conditions in the ghd7 prr37 double mutant [20]. This suggests that Hd1 functions in conjunction with Ghd7 and PRR37 under long-day conditions [21], while the OsELF4s-OsELF3-1-OsLUX complex reduces the expression of Ghd7 and PRR37 under short-day conditions. Therefore, Hd1 plays an independent role in the regulation of flowering [22]. Interestingly, it has been reported that Ghd7 and PRR37 are non-functional or exhibit weak functionality in the LJ11 background [23]. Therefore, knocking out Hd1 in the LJ11 background results in a late flowering phenotype.
As bbx2 flowered at a similar time to LJ11, this was probably due to functional redundancy with other BBX family members. To explore this, we investigated the evolutionary relationships within the BBX gene family. We identified BBX21 as a homolog of BBX2; however, its role in regulating rice flowering remains unknown. Future research will involve creating a bbx2 bbx21 double mutant to assess potential functional redundancy.
In summary, our study reveals that BBX2 functions as a negative regulator of flowering, and the expressions of Ehd1, Hd3a, and RFT1 are significantly reduced in BBX2 OE plants. BBX2 exhibits transcriptional repression activity and can interact with Hd1. These results suggest that BBX2 interacts with Hd1, co-suppressing the expression of Hd3a and consequently delaying flowering in rice.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14010048/s1. Supplemental Figure S1: Characterization of BBX2 OE plants in the LJ11 background. Supplemental Figure S2: Expression profiles of BBX2. Supplemental Figure S3: Characterization of bbx2, hd1, and bbx2 hd1 mutants in LJ11 background. Supplemental Table S1: Primers used in this study. Refs. [24,25] was cited in the Supplementary Materials.

Author Contributions

Q.B. and X.L. designed and supervised the research. Y.Y. and J.W. performed experiments, including material planting and the heading time survey. X.T. and C.L. analyzed the data. X.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Youth Innovation Promotion Association CAS (Grant No. 2022231), the Natural Science Foundation of Heilongjiang (YQ2022C039), and the Young Scientist Group Project of Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences (2023QNXZ02).

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary Materials.

Acknowledgments

We thank Yaoguang Liu for providing the vector pYLCRISPR/Cas9Pubi-H.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. OsBBX2 delays flowering by repressing Hd3a expression. (A) Representative image of LJ11 and BBX OE plants grown under LD conditions at the heading stage. (B) Flowering time of LJ11 and BBX OE under LD conditions. Data are means ± standard error (SE; n = 20). p values were calculated by Student’s t test compared to LJ11; **: p < 0.01. (C) Schematic diagrams of the reporter plasmids used in the rice protoplast transient assay. REN, Renilla luciferase; LUC, firefly luciferase. (D) The LUC activity in rice protoplasts with indicated reporter plasmids. Data are means ± SE (n = 3). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test). (E) The yeast two-hybrid assay showed that BBX2 interacts with Hd1. Yeast grew at 30 °C for 3 days. Empty vectors were used as the negative controls. AD, activation domain. BD, binding domain. (F) The LCI assay of the BBX2 interaction with Hd1 in N. benthamiana leaves. The co-transformation of cLUC-Hd1 and nLUC-BBX2 led to the re-constitution of the LUC signal, whereas no signal was detected when cLUC-Hd1 and nLUC, cLUC and nLUC-BBX2, and cLUC and nLUC were co-expressed. In each experiment, at least five independent N. benthamiana leaves were infiltrated and analyzed. (GI) A representative image of LJ11, bbx2 (G), hd1 (H), and bbx2 hd1 (I) mutants grown under the LD condition at the heading stage. (J) The flowering time of LJ11, bbx2, hd1, and bbx2 hd1 mutants under LD conditions. Data are means ± standard error (SE; n = 20). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test). (K) Schematic diagrams of the reporter plasmids used in the rice protoplast transient assay. 35SPro:GFP was used as the control and 35SPro:BBX2, 35SPro:Hd1, and Hd3aPro:LUC were used as the effectors and reporters. (L) Relative LUC activity expressed with reporters and effectors. The expression level of Renilla (REN) was used as an internal control. The LUC/REN ratio represents the relative activity of the Hd3a promoter. Data are shown as means ± SE (n = 3). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test).
Figure 1. OsBBX2 delays flowering by repressing Hd3a expression. (A) Representative image of LJ11 and BBX OE plants grown under LD conditions at the heading stage. (B) Flowering time of LJ11 and BBX OE under LD conditions. Data are means ± standard error (SE; n = 20). p values were calculated by Student’s t test compared to LJ11; **: p < 0.01. (C) Schematic diagrams of the reporter plasmids used in the rice protoplast transient assay. REN, Renilla luciferase; LUC, firefly luciferase. (D) The LUC activity in rice protoplasts with indicated reporter plasmids. Data are means ± SE (n = 3). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test). (E) The yeast two-hybrid assay showed that BBX2 interacts with Hd1. Yeast grew at 30 °C for 3 days. Empty vectors were used as the negative controls. AD, activation domain. BD, binding domain. (F) The LCI assay of the BBX2 interaction with Hd1 in N. benthamiana leaves. The co-transformation of cLUC-Hd1 and nLUC-BBX2 led to the re-constitution of the LUC signal, whereas no signal was detected when cLUC-Hd1 and nLUC, cLUC and nLUC-BBX2, and cLUC and nLUC were co-expressed. In each experiment, at least five independent N. benthamiana leaves were infiltrated and analyzed. (GI) A representative image of LJ11, bbx2 (G), hd1 (H), and bbx2 hd1 (I) mutants grown under the LD condition at the heading stage. (J) The flowering time of LJ11, bbx2, hd1, and bbx2 hd1 mutants under LD conditions. Data are means ± standard error (SE; n = 20). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test). (K) Schematic diagrams of the reporter plasmids used in the rice protoplast transient assay. 35SPro:GFP was used as the control and 35SPro:BBX2, 35SPro:Hd1, and Hd3aPro:LUC were used as the effectors and reporters. (L) Relative LUC activity expressed with reporters and effectors. The expression level of Renilla (REN) was used as an internal control. The LUC/REN ratio represents the relative activity of the Hd3a promoter. Data are shown as means ± SE (n = 3). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test).
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Yang, Y.; Wei, J.; Tian, X.; Liu, C.; Li, X.; Bu, Q. OsBBX2 Delays Flowering by Repressing Hd3a Expression Under Long-Day Conditions in Rice. Plants 2025, 14, 48. https://doi.org/10.3390/plants14010048

AMA Style

Yang Y, Wei J, Tian X, Liu C, Li X, Bu Q. OsBBX2 Delays Flowering by Repressing Hd3a Expression Under Long-Day Conditions in Rice. Plants. 2025; 14(1):48. https://doi.org/10.3390/plants14010048

Chicago/Turabian Style

Yang, Yusi, Jiaming Wei, Xiaojie Tian, Changhua Liu, Xiufeng Li, and Qingyun Bu. 2025. "OsBBX2 Delays Flowering by Repressing Hd3a Expression Under Long-Day Conditions in Rice" Plants 14, no. 1: 48. https://doi.org/10.3390/plants14010048

APA Style

Yang, Y., Wei, J., Tian, X., Liu, C., Li, X., & Bu, Q. (2025). OsBBX2 Delays Flowering by Repressing Hd3a Expression Under Long-Day Conditions in Rice. Plants, 14(1), 48. https://doi.org/10.3390/plants14010048

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