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Article

The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice

by
Yuan Liu
†,
Hongwanjun Zhang
†,
Wenqi Feng
,
Xiaolong Lin
,
Aijun Gao
,
Yang Cao
,
Qingqing Yang
,
Yingge Wang
,
Wanchen Li
,
Fengling Fu
and
Haoqiang Yu
*
Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region; Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Plants 2023, 12(16), 2995; https://doi.org/10.3390/plants12162995
Submission received: 18 July 2023 / Revised: 9 August 2023 / Accepted: 16 August 2023 / Published: 19 August 2023
(This article belongs to the Special Issue Molecular Biology of Plant Growth and Development)
Browse Figures
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Abstract

:
In model plants, the BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors play vital roles in regulating growth, development, and stimuli response. However, the roles of maize ZmBES1/BZR1 members are largely unknown. In this research, the ZmBES1/BZR1-9 gene was ectopically expressed in Arabidopsis and rice for the phenotyping of flowering. We found that the complementation and overexpression of ZmBES1/BZR1-9 in bes1-D mutant and wild type Arabidopsis both resulted in early flowering that was about 10 days shorter than in the untransformed control under long-day conditions. In addition, there was no difference in the rosette leaf number between all transgenic lines and the control. Subsequently, the ZmBES1/BZR1-9 gene was overexpressed in rice. It was found that overexpression lines of rice exhibited early flowering with heading dates that were 8 days shorter compared with untransformed plants. Moreover, the results of RNA-seq and qRT-PCR showed that five flowering-regulated genes, namely At2-MMP, AtPCC1, AtMYB56, AtPELPK1, and AtPRP10, were significantly up-regulated in all complementary and overexpressing lines of Arabidopsis. Meanwhile, the results of RNA-seq showed that 69 and 33 differentially expressed genes (DEGs) were up- and down-regulated in transgenic rice, respectively. Four flowering-related genes, namely OsGA20OX1, OsCCR19, OsBTBN19, and OsRNS4 were significantly up-regulated in transgenic lines. To sum up, our findings demonstrate that ZmBES1/BZR1-9 is involved in controlling flowering and provide insights into further underlying roles of BES1/BZR1s in regulating growth and development in crops.

1. Introduction

In higher plants, flowering is an important developmental process and marks the transition from vegetative growth to reproductive growth [1]. The photoperiod, vernalization, autonomic, gibberellin, age, and temperature pathways are among the main flowering-regulatory cues in plants. But, as crucial steroidal hormones, brassinosteroids (BRs) have been confirmed to control various biological processes in plants, such as growth and development regulation as well as stress resistance [2,3]. The BR signal is perceived by the receptor BRI1 and the co-receptor BAK1 on the cell membrane and triggers the phosphorylation and dephosphorylation of various downstream proteins, including BSKs (BR SIGNALING KINASES), CDGl (CONSTITUTIVE DIFFERENTIAL GROWTH1F), BSU1 (BRI1-SUPPRESSORF), BIN2 (BR-INSENSITIVE 2) [4,5,6,7]. Importantly, the inactivated BIN2 cannot phosphorylate the BES1 (BRI1-EMS-SUPPRESSOR 1) and BZR1 (BRASSINAZOLE RESISTANT 1) transcription factors, which also can be dephosphorylated by PP2A [6,8,9]. Thereafter, the activated BES1/BZR1s accumulate in the nucleus and regulate the transcription of nuclear genes by directly binding to elements of the E-box (CANNTG) or BRREs (CGTGT/CG) in gene promoters to regulate plant growth and BR synthesis [10,11,12,13].
As core transcription factors, previous studies have shown that BZR1/BES1s not only meditate BR signaling but also cross-talk with other hormone signals to regulate plant growth and stress resistance [14,15]. For instance, BES1/BZR1 can inhibit BR synthesis by binding to the BR synthesis genes promoter of DWF4 and CPD to inhibit their expression [10,12,16]. BES1 hinders ABI5 (ABSCISIC ACID INSENSITIVE5) to promote seed germination via ABA signaling and involves light to regulate plant growth mediated by the SINAT E3 ligase [17,18]. Exogenous GA can activate the BZR1-mediated BR signaling pathway [15]. Likewise, BES1/BZR1s regulate the adaptation to freezing, heat, phosphate-deficient, drought, and salt stress by up-regulating the expression of multiple stress-related genes in Arabidopsis, wheat, maize, tomato, and apple [19,20,21,22,23,24,25,26,27,28].
Moreover, BES1/BZR1s also recruit other regulators to regulate plant developmental processes. In Arabidopsis, dephosphorylated BES1 interacts with the UV receptor UVR8 and the blue light receptors CRY1 and CRY2 to regulate plant photomorphogenesis by inhibiting the binding between BES1 and DNA [29]. BES1 interacts with ERF6 (Early flowering 6) and its homolog REF6 to regulate target gene expression and to control flowering time [30]. BZR1 interacts with the cyclophilin Cyp20-2 to co-regulate flowering by suppressing FLD (FLOWERING LOCUS D) expression [31]. BZR1, together with BES1-INTERACTING MYC-like protein (BIM), recruits the H3K27 (histone 3 lysine 27) demethylase to activate FLC expression, leading to the inhibition of the flower transition [32]. Recently, it has been reported that BES1 acts on BEE1 (BR ENHANCED EXPRESSION 1), which directly binds to FT (FLOWERING LOCUS T), to control photoperiodic flowering [33]. In rice, OsBZR1 targets and interacts with histone deacetylase HDA703 to control rice growth and the heading date by repressing Ghd7 (HEADING DATE 7) expression [34]. However, the function of BES1/BZR1s is largely unknown in crops.
In maize, a total of eleven ZmBES1/BZR1 genes were identified in the maize genome in our previous study and by another group [35,36]. Subsequently, we confirmed that ZmBES1/BZR1-3 and -9 negatively regulates drought tolerance, while ZmBES1/BZR1-5 positively regulates salt and drought tolerance, the ABA response, and seed development [21,25,37]. Furthermore, ZmBES1/BZR1-2 has also been proven to positively regulate seed size and to promote the enlargement of the cotyledon and rosette leaves [38]. These reports suggest that ZmBES1/BZR1s members act on plant growth and development with functional diversity. However, whether ZmBES1/BZR1s is involved in flowering regulation is unknown. In this study, the function of ZmBES1/BZR1-9 in regulating flowering was evaluated by the heterologous expression of ZmBES1/BZR1-9 in Arabidopsis and rice. The study proves that ZmBES1/BZR1-9 is an activator of flowering and will provide insights into further underlying roles of BES1/BZR1s in regulating growth and development in crops.

2. Results

2.1. Generation of Transgenic Lines

To identify the function of ZmBES1/BZR1-9, it was transformed into Arabidopsis and rice, respectively. The positive transformants of Arabidopsis showed normal growth and robust green plants on plates with 50 mg/L kanamycin. In total, six homozygous lines were generated as identified by PCR amplification and reverse transcription PCR (RT-PCR), and these were named OE9-1, 9-2, 9-3, 9-5, 9-10, and 9-14. In these lines, the ZmBES1/BZR1-9 gene was successfully amplified from their genomic DNA (gDNA) and cDNA, respectively. However, there was no amplicon in the wild type (WT) (Figure 1). Meanwhile, five homozygous lines of rice expressing ZmBES1/BZR1-9 were produced and defined as R9-1, 9-2, 9-3, 9-4, and 9-5. These lines exhibited green leaves after soaking in 100 mg/L hygromycin solution, and the insertion of ZmBES1/BZR1-9 in the rice genome was confirmed by PCR and RT-PCR, respectively (Figure 2). The complementary lines L9-3 and L9-5 of the Arabidopsis mutant were produced in our previous study [21]. Hence, the complementary Arabidopsis lines L9-3 and 9-5, the overexpressing Arabidopsis lines OE9-2 and OE9-3, as well as the overexpressing rice lines R9-1 and R9-5 were used for phenotyping.

2.2. Expression of ZmBES1/BZR1-9 Accelerates Flowering in Arabidopsis

To explore the function of ZmBES1/BZR1-9 in the floral transition, the days from germination to flowering (DTF) of the complementary lines L9-3 and L9-5 were measured. As shown in Figure 3, under long-day (LD) conditions, the L9-3 and L9-5 lines exhibited early flowering and a significantly higher rate of flowering plants in the same growth stage compared with the bes1-D mutant, which showed delayed flowering compared with the WT. However, there was no difference in the total rosette leaf number (RLN) between the bes1-D, L9-3, and L9-5 lines. Meanwhile, the RLN of bes1-D was significantly higher than that of WT.
Meanwhile, to validate the early-flowering phenotype regulated by ZmBES1/BZR1-9, we generated the overexpressed lines OE9-2 and OE9-3 and conducted a phenotype characterization as a complementary assay. Similarly, early flowering was also observed in overexpressing lines under LD conditions. The DTF of the OE9-2 and OE9-3 lines was about 10 days earlier than that of WT. Compared with WT, the number of flowering plants in OE9-2 and OE9-3 was significantly higher from 55 days after sowing (Figure 4). At the same time, there was also no significant difference in RLN among different lines.
The above results indicate that the heterologous expression of the ZmBES1/BZR1-9 gene accelerates floral transition but does not affect the vegetative growth in Arabidopsis.
Moreover, in order to determine whether ZmBES1/BZR1-9 promotes flowering via the photoperiod pathway, L9-3, L9-5, and bes1-D as well as OE9-2, OE9-3 and WT were cultured under short-day (SD) conditions for phenotyping. We found that L9-3, L9-5, and bes1-D showed no bolting, with a severe vegetative growth phenotype at 125 days after sowing under SD conditions (Figure 5a). Likewise, although OE9-2, OE9-3, and WT gradually blossomed after 95 days of planting, there was no difference in flowering time among these lines (Figure 5b). The RLN also showed no difference (Figure 5c).

2.3. ZmBES1/BZR1-9 Promotes the Expression of Flowering-Related Genes in Arabidopsis

RNA-sequencing (RNA-seq) was performed in our previous study to interrogate the transcriptomic changes caused by ZmBES1/BZR1-9 in the L9-3 and L9-5 lines [21]. To investigate the potential mechanism of ZmBES1/BZR1-9 in controlling flowering in transgenic lines, the differentially expressed genes (DEGs) related to flowering were explored. In comparison with the bes1-D mutant, five DEGs associated with the regulation of flowering were identified in both the L9-3 and L9-5 lines, namely At2-MMP (AT1G70170), AtPCC1 (AT3G22231), AtMYB56 (AT5G17800), AtGRDP2 (AT4G37900), and AtPELPK1 (AT5G09530). Therefore, quantitative real-time PCR (RT-qPCR) was performed to analyze the expression of these genes in complementary lines, overexpressed lines, and an untransformed control. As shown in Figure 6, the expression of At2-MMP, AtPCC1, AtMYB56, AtGRDP2, and AtPELPK1 genes was significantly up-regulated in L9-3 and L9-5, as well as in OE9-2 and OE9-3, compared with bes1-D and WT, respectively. Meanwhile, 8, 10, 8, 13, and 16 E-box elements were found in the At2-MMP, AtPCC1, AtMYB56, AtGRDP2, and AtPELPK1 promoter regions, respectively (Table S2). The result indicates that ZmBES1/BZR1-9 promotes the flowering of transgenic Arabidopsis by up-regulating the expression of these genes.

2.4. ZmBES1/BZR1-9 Accelerates Flowering in Rice

To further analyze the role of ZmBES1/BZR1-9 in regulating flowering, the transgenic rice lines R9-1, R9-5, and the WT line were grown in Chengdu in summer (LD) and in Sanya in winter (SD). The results of phenotyping showed that there was no difference in heading date between the transgenic lines and the WT under LD conditions in summer. In contrast, compared with the WT, the transgenic lines R9-1 and R9-5 exhibited an earlier heading date and their flowering time was shortened by about 8 days under SD conditions in winter (Figure 7).

2.5. ZmBES1/BZR1-9 Regulates the Expression of Flowering-Associated GENES in Rice

RNA-seq was also conducted to analyze the DEGs regulated by ZmBES1/BZR1-9 in transgenic rice. As shown in Figure 8, compared with WT, a total of 102 common DEGs were identified and shared by the R9-1 and R9-5 lines. Among them, 67.65% of DEGs (69) were up-regulated, and 32.35% of DEGs (33) were down-regulated in the two transgenic lines. Gene Ontology (GO) analysis suggested that five DEGs were associated with flowering and involved in short-day photoperiodism and circadian rhythm; these were OsGA20OX1 (Os03g0856700), OsCCR19 (Os09g0419200), OsBTBN19 (Os09g0420900), OsRNS4 (Os09g0537700), and an unknown gene (Os02g0205500). Similarly, 15, 8, 14, 13, and 8 E-box elements were also found in the OsGA20OX1, OsCCR19, OsBTBN19, OsRNS4, and Os02g0205500 gene promoters, respectively (Table S2).

3. Discussion

Previous studies show that BR signaling is an important pathway involved in plant-flowering regulation. For instance, BR-insensitive and deficient mutants exhibit delayed flowering morphology, confirming that the components of the BR signal control the flowering transition [30,31,39,40,41]. BES1/BZR1s are key hubs in the BR signal [10,13]. In the present study, we found that the complementation and overexpression of the ZmBES1/BZR1-9 gene in WT and mutant Arabidopsis resulted in early flowering under LD conditions compared with the control (Figure 3 and Figure 4). Nevertheless, there was no significant difference in flowering time between the transgenic Arabidopsis and the control, which both exhibited exuberant vegetative growth of each line under SD conditions (Figure 5). The RLN is a key indicator of Arabidopsis flowering [42], but the transgenic plants showed no difference in RLN under LD and SD conditions (Figure 5). Meanwhile, transgenic rice exhibited a shorter heading date and about 8 days earlier flowering than WT under SD conditions, but there was no difference under natural LD conditions (Figure 7). This indicates that the ZmBES1/BZR1-9 gene positively regulates flowering but does not affect the vegetative growth process.
It has been confirmed that BES1 acts as a positive regulator of photoperiodic flowering in Arabidopsis [33]. As previously reported, the overexpression of BES1 promotes flowering, and its mutant and RNAi plants delayed flowering and possess significantly higher RLN than WT under LD conditions [32,33,43]. Similarly, overexpression of MiRZFP34 resulted in early flowering in transgenic Arabidopsis but no difference in RLN was observed [44]. Likewise, it was previously proven that histone deacetylase HDA703 interacts with OsBZR1 to control rice growth and heading by inhibiting Ghd7 expression [34]. Hence, it is speculated that the different phenotypes of transgenic Arabidopsis and rice expressing ZmBES1/BZR1-9 under LD and SD conditions may be due to the Arabidopsis being an LD plant but the rice being an SD plant [42]. In Arabidopsis, under LD conditions, the CO (CONSTANS) protein accumulated and induced the expression of FT and its homolog TSF (TWIN SISTER OF FT) to promote flowering [45,46]. However, the CO-homologous Hd1 promotes early heading by up-regulating the expression of the FT-homologous Hd3a gene under SD conditions in rice [47,48]. Therefore, we speculate that the ZmBES1/BZR1-9 gene regulates flowering through different photoperiod-mediated pathways in Arabidopsis and rice.
Furthermore, we showed that ZmBES1/BZR1-9 up-regulated the expression of At2-MMP, AtPCC1, AtMYB56, AtGRDP2, and AtPELPK1, as well as of OsGA20OX1, OsCCR19, OsBTBN19, and OsRNS4, in transgenic Arabidopsis and rice, respectively (Figure 6 and Figure 8). This can be explained by the binding of ZmBES1/BZR1-9 to E-box elements in these genes promoters to promote their expression (Table S2) because the ZmBES1/BZR1-9 protein localizes in the nucleus and functions as a transcription factor [21]. It was confirmed that BES1/BZR1s can directly bind to E-box or BRRE elements to regulate the transcription of target genes [10,13]. Previous studies confirmed that at2-mmp mutant, AtPCC1 and AtPELPK1 RNAi plants, and AtGRDP2 knockout lines showed delayed flowering, suggesting their positive roles in regulating flowering [49,50,51,52]. Likewise, PPC1 exhibits a circadian-regulated expression pattern, is involved in light-regulated development via interaction with the COP9 signalosome subunit 5, and regulates the flowering transition in the photoperiod-dependent pathway [52,53,54]. AtGRDP2 also regulates female gametophyte development via the auxin pathway, and plants overexpressing it show early flowering [50,55]. Meanwhile, BES1 directly represses AtMYB56, which positively regulates the quiescent center and cell division [56,57]. In rice, OsGA20OX1 influences GA levels, is expressed in reproductive meristems, and crosstalks with cytokinin to regulate growth and development [58,59]. GA also regulates flowering [60,61]. OsRNS4 is regulated by phytochrome (pyh) A-, B-, and C-mediated light signals in rice [62]. It has been shown that phyA, B, and C play crucial roles in plant flowering [63,64,65,66]. In addition, OsBTBN19 and its homolog, NPY1, encode BTB domain proteins, which are reported to regulate flowering [67,68], although the OsBTBN19 and OsCCR19 genes were not directly confirmed to regulate flowering.
The study suggests that the ZmBES1/BZR1-9 gene positively regulates flowering in transgenic Arabidopsis and rice and that its overexpression can be used to shorten the flowering period. Meanwhile, the mechanism of ZmBES1/BZR1-9 in regulating flowering in maize is unknown and will be revealed in our future studies.

4. Materials and Methods

4.1. Plants Materials and Growth Conditions

Arabidopsis thaliana (Col-0) and Oryza sativa (Nipponbare) were used for the overexpression of the ZmBES1/BZR1-9 gene. The complementary lines (L9-3 and L9-5) of the Arabidopsis bes1-D mutant were previously produced in our laboratory [21]. All Arabidopsis plants were grown in the growth chambers under an artificial LD photoperiod (14 h light/10 h dark, LD) or SD photoperiod (10 h light/14 h dark, SD) under 60–70% relative humidity at 22 °C. The rice seedlings were grown in the field in Chengdu in summer (from mid-May to mid-July; LD conditions with 13.4–14 h of light) and in Sanya in winter (from mid-Oct to mid-next March; natural SD conditions with 9.5–12 h light).

4.2. Vector Construction and Transformation

The specific primers 1300-F and 1300-R (Table S1) were designed using Primer5.0, synthesized at Sangon Biotech (Shanghai, China), and used to amplify the coding sequence (CDS) of the ZmBES1/BZR1-9 gene from the 35S::ZmBES1/BZR1-9-eGFP plasmid constructed previously [21]. The PCR products and the pCAMBIA1300 plasmid were digested using Hind III and BamH I. After digestion, the PCR products were subsequently cloned into Hind III and BamH I sites of the pCAMBIA1300 plasmid to generate 35S::ZmBES1/BZR1-9 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The ZmBES1/BZR1-9 gene was driven by the 35S promoter and terminated by CaMV Poly(A).
The 35S::ZmBES1/BZR1-9 plasmids were introduced into the Agrobacterium tumefaciens strain GV3101 for the next transformation. To create overexpressed lines of Arabidopsis, the transformation of Arabidopsis and rice was performed by the floral dip method and Agrobacterium-mediated calli transformation, respectively [69,70]. After transformation, the seeds of transgenic Arabidopsis were screened using 50 mg/L kanamycin (Sigma, St. Louis, MI, USA) to screening for transformants. The transgenic rice plants were screened by using 100 ng/mL hygromycin B (Coolaber, Beijing, China). The positive Arabidopsis seedlings with kanamycin resistance and positive rice seedlings with hygromycin resistance were harvested individually. In the T2 generation, the plants showed a 3:1 segregation for resistance/susceptibility to kanamycin or hygromycin and were self-pollinated to generate T3. Then, the seeds of each line were screened using the same methods. The homozygous lines were identified without segregation and used in the next study.

4.3. PCR and RT-PCR

The gDNA of the transgenic lines and the WT line was extracted using the CTAB method [71]. To confirm the insertion of ZmBES1/BZR1-9 in the genome of Arabidopsis and rice, a pair of specific primers, 9F and 9R, were designed, synthesized, and used to amplify a 911 bp fragment from ZmBES1/BZR1-9. Moreover, the total RNA of each line was extracted, and the gDNA was removed and reverse-transcribed to cDNA using the RNAiso Plus kit (Takara) and HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), respectively. The RT-PCR was performed to detect the transcription of the ZmBES1/BZR1-9 gene in transgenic lines. Meanwhile, the primer pairs qAf/qAr and qGf/qGr were designed, synthesized, and used to amplify AtACTIN2 and OsGAPDH genes, respectively, which were used as the internal controls. The sequences of primers used for RT-PCR are also listed in Table S1.

4.4. Phenotyping of Transgenic Lines

To analyze the flowering time of transgenic Arabidopsis, the seeds of the complementary lines, overexpressed lines, WT, and bes1-D mutant were sown in soil and cultured in the growth chambers under the conditions described above. The days from germination to flowering, the total rosette leaf number, and the percentage of flowering plants over the same time period were measured from 25 plants and used to monitor the flowering time, as described by Li et al. [32] and Wang et al. [33]. For the phenotyping of transgenic rice, the days from seed germination to the appearance of the first main panicle were counted and used to detect the heading date, as described by Lu et al. [44]. In each replicate, 20 plants of every line were scored. Three replicates of each experiment were performed in this study. All statistical data were calculated using GraphPad Prism and Microsoft Excel 2017 and were presented as the mean ± SE. Student’s t-tests were used to analyze the significance of the data between the transgenic lines and the WT. * and ** represent p < 0.05 and <0.01, respectively.

4.5. RNA-Seq Analysis

The RNA-seq analysis was conducted as our previous study [21]. In brief, the total RNA was extracted from two-week-old seedlings of R9-1, R9-2, and WT using the RNAprep Pure Plant Kit. Then, each RNA sample was qualified by testing their quality and integrity and used for sequencing library preparation using the Bioanalyzer 2100 and the VAHTSTM mRNA-seq V2 Library Prep Kit, respectively. The library was sequenced at the Sanshu Biotechnology Company (Shanghai, China) using the Novaseq 6000 system. The sequencing data were analyzed as described by Sun et al. [25]. The raw data were evaluated and filtered by removing sequencing adapters and low-quality reads as well as contaminants to produce clean data using FastQC (version 0.11.2) and Trimmomatic (version 0.36). Then, clean data were aligned with the Arabidopsis genome (TAIR 10) using Hisat2 and used to assemble transcripts of each gene using StringTie. The read counts of each gene assembled by StringTie were used to identify DEGs using DESeq2 with a p-value < 0.05 and |FoldChange| > 2. The GO enrichment analysis was performed using KOBAS accessed on 15 February 2023 (http://kobas.cbi.pku.edu.cn/anno_iden.php).

4.6. qRT-PCR

The expression of candidate genes in transgenic lines was analyzed by qRT-PCR using PerfectStart®Green qPCR SuperMix (TransGen, Beijing, China) in the CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA), as described in our previous study [25]. The procedure of qRT-PCR consisted of a two-step temperature cycle with pre-degeneration at 95 ℃ for 30 s, 39 cycles of degeneration at 95 for 5 s, and an extension step at 58 °C for 30 s. The temperature was set to increase to 95 ℃ by 0.5 ℃/s at the end of each last cycle to differentiate between specific and non-specific amplicons. Likewise, the AtACTIN2 and OsGAPDH genes were amplified using the primer pairs qAf/qAr and qGf/qGr, respectively, and used as the internal controls. The relative expression level was normalized following the 2−ΔΔCt method [72]. The sequences of these candidates were derived from the National Center for Biotechnology Information (NCBI) or the Arabidopsis Information Resource (TAIR) and used to design primers using primer-BLAST accessed on 5 November 2021 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). The primers are listed in Table S1. The candidate genes include AtACTIN2 (AT3G18780), OsGAPDH (Os04g40950), At2-MMP (AT1G70170), AtPCC1 (AT3G22231), AtMYB56 (AT5G17800), AtGRDP2 (AT4G37900), AtPELPK1 (AT5G09530), OsCCR19 (Os09g0419200), OsBNTB19 (Os09g0420900), OsRNS4 (Os09g0537700), and OsGA20OX1 (Os03g0856700).

5. Conclusions

In conclusion, the objective of the study was to validate the role of the maize ZmBES1/BZR1-9 transcription factor in regulating flowering time. The ZmBES1/BZR1-9 gene was ectopically expressed in Arabidopsis and rice for phenotyping. Our findings demonstrate that ZmBES1/BZR1-9 is a positive regulator that promotes flowering via multiple photoperiod-mediated pathways, but it does not affect vegetative growth. Our results also suggest that the maize ZmBES1/BZR1-9 gene can be used to improve the flowering period via its overexpression and provide a reference for further underlying roles of BES1/BZR1 in regulating growth and development in crops.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12162995/s1, Table S1: Primers used in the study; Table S2: The number of E-box and BRRE elements in the promoter region of the candidate gene.

Author Contributions

Conceptualization, Y.L. and H.Y.; methodology, Y.L., H.Z., W.F., X.L., A.G. and Y.C.; software, Y.L.; formal analysis, Y.L. and H.Z.; data curation, Y.L.; writing—original draft preparation, Y.L.; writing—review and editing, W.L., F.F. and H.Y.; supervision, Q.Y. and Y.W.; project administration, H.Y.; funding acquisition, Y.W. and H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Sichuan Science and Technology Program (2022YFH0067) and the National Natural Science Foundation of China (32102226).

Data Availability Statement

All data are included in the article and Supplementary Files.

Acknowledgments

The authors thank the technical support team from the Key Laboratory of Biology and Genetic Improvement of Maize in the Southwest Region.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kobayashi, Y.; Weigel, D. Move on up, it’s time for change-mobile signals controlling photoperiod-dependent flowering. Genes Dev. 2007, 21, 2371–2384. [Google Scholar] [CrossRef]
  2. Clouse, S.D.; Sasse, J.M. BRASSINOSTEROIDS: Essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998, 49, 427–451. [Google Scholar] [CrossRef]
  3. Mitchell, J.W.; Mandava, N.; Worley, J.F.; Plimmer, J.R.; Smith, M.V. Brassins-a new family of plant hormones from rape pollen. Nature 1970, 225, 1065–1066. [Google Scholar] [CrossRef]
  4. Kim, T.W.; Guan, S.; Burlingame, A.L.; Wang, Z.Y. The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2. Mol. Cell 2011, 43, 561–571. [Google Scholar] [CrossRef] [PubMed]
  5. Sun, Y.; Han, Z.; Tang, J.; Hu, Z.; Chai, C.; Zhou, B.; Chai, J. Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res. 2013, 23, 1326–1329. [Google Scholar] [CrossRef] [PubMed]
  6. Tang, W.; Kim, T.W.; Oses-Prieto, J.A.; Sun, Y.; Deng, Z.; Zhu, S.; Wang, R.; Burlingame, A.L.; Wang, Z.Y. BSKs mediate signal transduction from the receptor Kinase BRI1 in Arabidopsis. Science 2008, 321, 557–560. [Google Scholar] [CrossRef]
  7. Wang, X.; Chory, J. Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 2006, 313, 1118–1122. [Google Scholar] [CrossRef] [PubMed]
  8. Kim, T.W.; Guan, S.; Sun, Y.; Deng, Z.; Tang, W.; Shang, J.X.; Sun, Y.; Burlingame, A.L.; Wang, Z.Y. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 2009, 11, 1254–1260. [Google Scholar] [CrossRef]
  9. Rubbo, S.D.; Irani, N.G.; Russinova, E. PP2A Phosphatases: The “On-Off” regulatory switches of brassinosteroid signaling. Sci. Signal. 2011, 4, 25. [Google Scholar] [CrossRef] [PubMed]
  10. He, J.X.; Gendron, J.M.; Sun, Y.; Gampala, S.S.; Gendron, N.; Sun, C.Q.; Wang, Z.Y. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 2005, 307, 1634–1638. [Google Scholar] [CrossRef]
  11. Ibanez, C.; Delker, C.; Martinez, C.; Burstenbinder, K.; Janitza, P.; Lippmann, R.; Ludwig, W.; Sun, H.; James, G.V.; Klecker, M.; et al. Brassinosteroids dominate hormonal regulation of plant thermosmorphogenesis via BZR1. Curr. Biol. 2018, 28, 303–310. [Google Scholar] [CrossRef] [PubMed]
  12. Tanaka, K.; Asami, T.; Yoshida, S.; Nakamura, Y.; Matsuo, T.; Okamoto, S. Brassinosteroid homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its metabolism. Plant Physiol. 2005, 138, 1117–1125. [Google Scholar] [CrossRef] [PubMed]
  13. Yin, Y.; Vafeados, D.; Tao, Y.; Yoshida, S.; Asami, T.; Chory, J. A new class of transcription factors mediates brassinosteroid regulated gene expression in Arabidopsis. Cell 2005, 120, 249–259. [Google Scholar] [CrossRef] [PubMed]
  14. Nolan, T.M.; Vukasinovic, N.; Liu, D.; Russinova, E.; Yin, Y. Brassinosteroids: Multidimensional regulators of plant growth, development, and stress responses. Plant Cell 2020, 32, 295–318. [Google Scholar] [CrossRef]
  15. Unterholzner, S.J.; Rozhon, W.; Papacek, M.; Ciomas, J.; Lange, T.; Kugler, K.G.; Mayer, K.F.; Sieberer, T.; Poppenberger, B. Brassinosteroids are master regulators of gibberellin biosynthesis in Arabidopsis. Plant Cell 2015, 27, 2261–2272. [Google Scholar] [CrossRef]
  16. Wang, Z.; Nakano, T.; Gendron, J.; He, J.; Chen, M.; Vafeados, D.; Yang, Y.; Fujioka, S.; Yoshida, S.; Asami, T.; et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev. Cell 2022, 2, 505–513. [Google Scholar] [CrossRef] [PubMed]
  17. Yang, M.; Li, C.; Cai, Z.; Hu, Y.; Nolan, T.; Yu, F.; Yin, Y.; Xie, Q.; Tang, G.; Wang, X. SINAT E3 ligases control the light-mediated stability of the brassinosteroid-activated transcription factor BES1 in Arabidopsis. Dev. Cell 2017, 41, 47–58. [Google Scholar] [CrossRef] [PubMed]
  18. Zhao, X.; Dou, L.; Gong, Z.; Wang, X.; Mao, T. BES1 hinders ABSCISIC ACID INSENSITIVE5 and promotes seed germination in Arabidopsis. New Phytol. 2019, 221, 908–918. [Google Scholar] [CrossRef]
  19. Albertos, P.; Dündar, G.; Schenk, P.; Carrera, S.; Cavelius, P.; Sieberer, T.; Poppenberger, B. Transcription factor BES1 interacts with HSFA1 to promote heat stress resistance of plants. EMBO J. 2022, 41, e108664. [Google Scholar] [CrossRef] [PubMed]
  20. Cui, X.Y.; Gao, Y.; Guo, J.; Yu, T.F.; Zheng, W.J.; Liu, Y.W.; Chen, J.; Xu, Z.S.; Ma, Y.Z. BES/BZR transcription factor TaBZR2 positively regulates drought responses by activation of TaGST1. Plant Physiol. 2019, 180, 605–620. [Google Scholar] [CrossRef] [PubMed]
  21. Feng, W.; Liu, Y.; Cao, Y.; Zhao, Y.; Zhang, H.; Sun, F.; Yang, Q.; Li, W.; Lu, Y.; Zhang, X.; et al. Maize ZmBES1/BZR1-3 and-9 transcription factors negatively regulate drought tolerance in transgenic Arabidopsis. Int. J. Mol. Sci. 2022, 23, 6025. [Google Scholar] [CrossRef] [PubMed]
  22. Jia, C.; Zhao, S.; Bao, T.; Zhao, P.; Peng, K.; Guo, Q.; Gao, X.; Qin, J. Tomato BZR/BES transcription factor SlBZR1 positively regulates BR signaling and salt stress tolerance in tomato and Arabidopsis. Plant Sci. 2021, 302, 110719. [Google Scholar] [CrossRef] [PubMed]
  23. Li, H.; Ye, K.; Shi, Y.; Cheng, J.; Zhang, X.; Yang, S. BZR1 positively regulates freezing tolerance via CBF-dependent and CBF-independent pathways in Arabidopsis. Mol. Plant 2017, 10, 545–559. [Google Scholar] [CrossRef]
  24. Singh, A.P.; Fridman, Y.; Friedlander-Shani, L.; Tarkowska, D.; Strnad, M.; Savaldi-Goldstein, S. Activity of the brassinosteroid transcription factors BRASSINAZOLE RESISTANT1 and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1/BRASSINAZOLE RESISTANT2 blocks developmental reprogramming in response to low phosphate availability. Plant Physiol. 2014, 166, 678–688. [Google Scholar] [CrossRef] [PubMed]
  25. Sun, F.; Yu, H.; Qu, J.; Cao, Y.; Ding, L.; Feng, W.; Khalid, M.H.B.; Li, W.; Fu, F. Maize ZmBES1/BZR1-5 decreases ABA sensitivity and confers tolerance to osmotic stress in transgenic Arabidopsis. Int. J. Mol. Sci. 2020, 21, 996. [Google Scholar] [CrossRef] [PubMed]
  26. Van Nguyen, T.; Park, C.R.; Lee, K.H.; Lee, S.; Kim, C.S. BES1/BZR1 homolog 3 cooperates with E3 ligase AtRZF1 to regulate osmotic stress and brassinosteroid responses in Arabidopsis. J. Exp. Bot. 2021, 72, 636–653. [Google Scholar] [CrossRef]
  27. Wang, X.; Chen, X.; Wang, Q.; Chen, M.; Liu, X.; Gao, D.; Li, D.; Li, L. MdBZR1 and MdBZR1-2 like transcription factors improves salt tolerance by regulating gibberellin biosynthesis in apple. Front. Plant Sci. 2019, 10, 1473. [Google Scholar] [CrossRef]
  28. Yin, Y.; Qin, K.; Song, X.; Zhang, Q.; Zhou, Y.; Xia, X.; Yu, J. BZR1 transcription factor regulates heat stress tolerance through FERONIA receptor-like kinase-mediated reactive oxygen species signaling in tomato. Plant Cell Physiol. 2018, 59, 2239–2254. [Google Scholar] [CrossRef]
  29. Wang, W.; Lu, X.; Li, L.; Lian, H.; Mao, Z.; Xu, P.; Guo, T.; Xu, F.; Du, S.; Cao, X.; et al. Photoexcited CRYPTOCHROME1 interacts with dephosphorylated BES1 to regulate brassinosteroid signaling and photomorphogenesis in Arabidopsis. Plant Cell 2018, 30, 1989–2005. [Google Scholar] [CrossRef]
  30. Yu, X.; Li, L.; Li, L.; Guo, M.; Chory, J.; Yin, Y. Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc. Natl. Acad. Sci. USA 2008, 105, 7618–7623. [Google Scholar] [CrossRef] [PubMed]
  31. Zhang, Y.; Li, B.; Xu, Y.; Li, H.; Li, S.; Zhang, D.; Mao, Z.; Guo, S.; Yang, C.; Weng, Y.; et al. The cyclophilin CYP20-2 modulates the conformation of BRASSINAZOLE-RESISTANT1, which binds the promoter of FLOWERING LOCUS D to regulate flowering in Arabidopsis. Plant Cell 2013, 25, 2504–2521. [Google Scholar] [CrossRef] [PubMed]
  32. Li, Z.; Ou, Y.; Zhang, Z.; Li, J.; He, Y. Brassinosteroid signaling recruits histone 3 lysine-27 demethylation activity to FLOWERING LOCUS C chromatin to inhibit the floral transition in Arabidopsis. Mol. Plant 2018, 11, 1135–1146. [Google Scholar] [CrossRef] [PubMed]
  33. Wang, F.; Gao, Y.; Liu, Y.; Zhang, X.; Gu, X.; Ma, D.; Zhao, Z.; Yuan, Z.; Xue, H.; Liu, H. BES1-regulated BEE1 controls photoperiodic flowering downstream of blue light signaling pathway in Arabidopsis. New Phytol. 2019, 223, 1407–1419. [Google Scholar] [CrossRef]
  34. Wang, H.; Jiao, X.; Kong, X.; Liu, Y.; Chen, X.; Fang, R.; Yan, Y. The histone deacetylase HDA703 interacts with OsBZR1 to regulate rice brassinosteroid signaling, growth and heading date through repression of Ghd7 expression. Plant J. 2020, 104, 447–459. [Google Scholar] [CrossRef]
  35. Manoli, A.; Trevisan, S.; Quaggiotti, S.; Varotto, S. Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L. Plant Growth Regul. 2018, 84, 423–436. [Google Scholar] [CrossRef]
  36. Yu, H.; Feng, W.; Sun, F.; Zhang, Y.; Qu, J.; Liu, B.; Lu, F.; Yang, L.; Fu, F.; Li, W. Cloning and characterization of BES1/BZR1 transcription factor genes in maize. Plant Growth Regul. 2018, 86, 235–249. [Google Scholar] [CrossRef]
  37. Sun, F.; Ding, L.; Feng, W.; Cao, Y.; Lu, F.; Yang, Q.; Li, W.; Lu, Y.; Shabek, N.; Fu, F.; et al. Maize transcription factor ZmBES1/BZR1-5 positively regulates kernel size. J. Exp. Bot. 2021, 72, 1714–1726. [Google Scholar] [CrossRef]
  38. Zhang, X.; Guo, W.; Du, D.; Pu, L.; Zhang, C. Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants. Plant Sci. 2020, 292, 110378. [Google Scholar] [CrossRef]
  39. Clouse, S.D. The molecular intersection of brassinosteroid regulated growth and flowering in Arabidopsis. Proc. Natl. Acad. Sci. USA 2008, 105, 7345–7346. [Google Scholar] [CrossRef]
  40. Domagalska, M.A.; Schomburg, F.M.; Amasino, R.M.; Vierstra, R.D.; Nagy, F.; Davis, S.J. Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering. Development 2007, 134, 2841–2850. [Google Scholar] [CrossRef]
  41. Zhao, B.; Li, J. Regulation of brassinosteroid biosynthesis and inactivation. J. Integr. Plant Biol. 2012, 54, 746–759. [Google Scholar] [CrossRef]
  42. Amasino, R. Seasonal and developmental timing of flowering. Plant J. 2010, 61, 1001–1013. [Google Scholar] [CrossRef] [PubMed]
  43. Jiang, J.; Zhang, C.; Wang, X. A recently evolved isoform of the transcription factor BES1 promotes brassinosteroid signaling and development in Arabidopsis thaliana. Plant Cell 2015, 27, 361–374. [Google Scholar] [CrossRef] [PubMed]
  44. Lu, S.; Zhang, N.; Xu, Y.; Chen, H.; Huang, J.; Zou, B. Functional conservation and divergence of MOS1 that controls flowering time and seed size in rice and Arabidopsis. Int. J. Mol. Sci. 2022, 23, 13448. [Google Scholar] [CrossRef]
  45. Bradley, D.; Ratcliffe, O.; Vincent, C.; Carpenter, R.; Coen, E. Inflorescence commitment and architecture in Arabidopsis. Science 1997, 275, 80–83. [Google Scholar] [CrossRef]
  46. Yamaguchi, A.; Kobayashi, Y.; Goto, K.; Abe, M.; Araki, T. TWIN SISTER OF FT (TSF) acts as a floral pathway integrator redundantly with FT. Plant Cell Physiol. 2005, 46, 1175–1189. [Google Scholar] [CrossRef]
  47. Kojima, S.; Takahashi, Y.; Kobayashi, Y.; Monna, L.; Sasaki, T.; Araki, T.; Yano, M. Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol. 2002, 43, 1096–1105. [Google Scholar] [CrossRef] [PubMed]
  48. Komiya, R.; Ikegami, A.; Tamaki, S.; Yokoi, S.; Shimamoto, K. Hd3a and RFT1 are essential for flowering in rice. Development 2008, 135, 767–774. [Google Scholar] [CrossRef]
  49. Golldack, D.; Popova, O.V.; Dietz, K.J. Mutation of the matrix metalloproteinase At2-MMP inhibits growth and causes late flowering and early senescence in Arabidopsis. J. Biol. Chem. 2002, 277, 5541–5547. [Google Scholar] [CrossRef] [PubMed]
  50. Ortega-Amaro, M.A.; Rodriguez-Hernandez, A.A.; Rodriguez-Kessler, M.; Hernandez-Lucero, E.; Rosales-Mendoza, S.; Ibanez-Salazar, A.; Delgado-Sanchez, P.; Jimenez-Bremont, J.F. Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance. Front. Plant Sci. 2015, 5, 782. [Google Scholar] [CrossRef] [PubMed]
  51. Rashid, A.; Deyholos, M.K. PELPK1 (At5g09530) contains a unique pentapeptide repeat and is a positive regulator of germination in Arabidopsis thaliana. Plant Cell Rep. 2011, 30, 1735–1745. [Google Scholar] [CrossRef] [PubMed]
  52. Segarra, S.; Mir, R.; Martinez, C.; Leon, J. Genome-wide analyses of the transcriptomes of salicylic acid-deficient versus wild-type plants uncover Pathogen and Circadian Controlled 1 (PCC1) as a regulator of flowering time in Arabidopsis. Plant Cell Environ. 2010, 33, 11–22. [Google Scholar] [CrossRef] [PubMed]
  53. Mir, R.; Leon, J. Pathogen and circadian controlled 1 (PCC1) protein is anchored to the plasma membrane and interacts with subunit 5 of COP9 signalosome in Arabidopsis. PLoS ONE 2014, 9, e87216. [Google Scholar] [CrossRef]
  54. Sauerbrunn, N.; Schlaich, N.L. PCC1: A merging point for pathogen defense and circadian signaling in Arabidopsis. Planta 2004, 218, 552–561. [Google Scholar] [CrossRef]
  55. Wang, L.; Liu, Y.; Aslam, M.; Jakada, B.H.; Qin, Y.; Cai, H. The Glycine-rich domain protein GRDP2 regulates ovule development via the auxin pathway in Arabidopsis. Front. Plant Sci. 2021, 12, 698487. [Google Scholar] [CrossRef] [PubMed]
  56. Vilarrasa-Blasi, J.; González-Garcia, M.P.; Frigola, D.; Fabregas, N.; Alexiou, K.G.; Lopez-Bigas, N.; Rivas, S.; Jauneau, A.; Lohmann, J.U.; Benfey, P.N.; et al. Regulation of plant stem cell quiescence by a brassinosteroid signaling module. Dev. Cell 2014, 30, 36–47. [Google Scholar] [CrossRef] [PubMed]
  57. Zhang, Y.; Liang, W.; Shi, J.; Xu, J.; Zhang, D. MYB56 encoding a R2R3 MYB transcription factor regulates seed size in Arabidopsis thaliana. J. Integr. Plant Biol. 2013, 55, 1166–1178. [Google Scholar] [CrossRef]
  58. Bolduc, N.; Hake, S. The maize transcription factor KNOTTED1 directly regulates the gibberellin catabolism gene ga2ox1. Plant Cell 2009, 21, 1647–1658. [Google Scholar] [CrossRef]
  59. Wu, Y.; Wang, Y.; Mi, X.F.; Shan, J.X.; Li, X.M.; Xu, J.L.; Lin, H.X. The QTL GNP1 encodes GA20ox1, which increases grain number and yield by increasing cytokinin activity in rice panicle meristems. PLoS Genet. 2016, 12, e1006386. [Google Scholar] [CrossRef]
  60. Conti, L. Hormonal control of the floral transition: Can one catch them all? Dev. Biol. 2017, 430, 288–301. [Google Scholar] [CrossRef]
  61. Wilson, R.N.; Heckman, J.W.; Somerville, C.R. Gibberellin is required for flowering in Arabidopsis thaliana under short days. Plant Physiol. 1992, 100, 403–408. [Google Scholar] [CrossRef] [PubMed]
  62. Zheng, J.; Wang, Y.; He, Y.; Zhou, J.; Li, Y.; Liu, Q.; Xie, X. Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice. Plant Sci. 2014, 214, 99–105. [Google Scholar] [CrossRef] [PubMed]
  63. Sun, W.; Xu, X.H.; Lu, X.; Xie, L.; Bai, B.; Zheng, C.; Sun, H.; He, Y.; Xie, X.Z. The rice phytochrome genes, PHYA and PHYB, have synergistic effects on anther development and pollen viability. Sci. Rep. 2017, 7, 6439. [Google Scholar] [CrossRef] [PubMed]
  64. Woods, D.P.; Li, W.; Sibout, R.; Shao, M.; Laudencia-Chingcuanco, D.; Vogel, J.P.; Dubcovsky, J.; Amasino, R.M. PHYTOCHROME C regulation of photoperiodic flowering via PHOTOPERIOD1 is mediated by EARLY FLOWERING 3 in Brachypodium distachyon. PLoS Genet. 2023, 19, e1010706. [Google Scholar] [CrossRef] [PubMed]
  65. Yuan, J.; Ott, T.; Hiltbrunner, A. Phytochromes and flowering: Legumes do it another way. Trends Plant Sci. 2023, 28, 379–381. [Google Scholar] [CrossRef] [PubMed]
  66. Zhao, F.; Lyu, X.; Ji, R.; Liu, J.; Zhao, T.; Li, H.; Liu, B.; Pei, Y. CRISPR/Cas9-engineered mutation to identify the roles of phytochromes in regulating photomorphogenesis and flowering time in soybean. Crop J. 2022, 10, 1654–1664. [Google Scholar] [CrossRef]
  67. Cheng, Y.; Qin, G.; Dai, X.; Zhao, Y. NPY1, a BTB-NPH3-like protein, plays a critical role in auxin-regulated organogenesis in Arabidopsis. Proc. Natl. Acad. Sci. USA 2007, 104, 18825–18829. [Google Scholar] [CrossRef] [PubMed]
  68. Shalmani, A.; Huang, Y.B.; Chen, Y.B.; Muhammad, I.; Li, B.B.; Ullah, U.; Jing, X.Q.; Bhanbhro, N.; Liu, W.T.; Li, W.Q.; et al. The highly interactive BTB domain targeting other functional domains to diversify the function of BTB proteins in rice growth and development. Int. J. Biol. Macromol. 2021, 192, 1311–1324. [Google Scholar] [CrossRef] [PubMed]
  69. Ali, I.; Sher, H.; Ali, A.; Hussain, S.; Ullah, Z. Simplified floral dip transformation method of Arabidopsis thaliana. J. Microbiol. Methods 2022, 197, 106492. [Google Scholar] [CrossRef] [PubMed]
  70. Hiei, Y.; Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 2008, 3, 824–834. [Google Scholar] [CrossRef] [PubMed]
  71. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytocheml. Bull. 1987, 19, 11–15. [Google Scholar]
  72. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Identification of transgenic Arabidopsis lines. (a) PCR detection. (b) RT-PCR. OE9-1, 9-2, 9-3, 9-5, 9-10, and 9-14 represent homozygous lines overexpressing the ZmBES1/BZR1-9 gene in Arabidopsis. The 911 bp fragment of ZmBES1/BZR1-9 was amplified and detected by PCR (a) and RT-PCR (b), respectively. M, DNA 2000 standard consisting of a 2000, 1000, 750, 500, 250, and 100 bp ladder from top to bottom. WT, wild type. The 545 bp fragment of AtACTIN2 was amplified and used as a reference.
Figure 1. Identification of transgenic Arabidopsis lines. (a) PCR detection. (b) RT-PCR. OE9-1, 9-2, 9-3, 9-5, 9-10, and 9-14 represent homozygous lines overexpressing the ZmBES1/BZR1-9 gene in Arabidopsis. The 911 bp fragment of ZmBES1/BZR1-9 was amplified and detected by PCR (a) and RT-PCR (b), respectively. M, DNA 2000 standard consisting of a 2000, 1000, 750, 500, 250, and 100 bp ladder from top to bottom. WT, wild type. The 545 bp fragment of AtACTIN2 was amplified and used as a reference.
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Plants 12 02995 g002
Figure 2. Screening of transgenic rice. (a) Antibiotic screening. (b) PCR amplification. (c) RT-PCR. R9-1, R9-2, R9-3, R9-4, and R9-5 mean transgenic rice lines expressing the ZmBES1/BZR1-9 gene. The 911 bp and 582 bp fragments of ZmBES1/BZR1-9 were amplified and detected by PCR and RT-PCR, respectively. M, DNA 2000 standard consisting of a 2000, 1000, 750, 500, 250, and 100 bp ladder from top to bottom. WT, wild type. The 629 bp fragment of the OsGAPDH gene was amplified and used as a reference.
Figure 2. Screening of transgenic rice. (a) Antibiotic screening. (b) PCR amplification. (c) RT-PCR. R9-1, R9-2, R9-3, R9-4, and R9-5 mean transgenic rice lines expressing the ZmBES1/BZR1-9 gene. The 911 bp and 582 bp fragments of ZmBES1/BZR1-9 were amplified and detected by PCR and RT-PCR, respectively. M, DNA 2000 standard consisting of a 2000, 1000, 750, 500, 250, and 100 bp ladder from top to bottom. WT, wild type. The 629 bp fragment of the OsGAPDH gene was amplified and used as a reference.
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Plants 12 02995 g003
Figure 3. The phenotype of complementary lines under LD conditions (14 h light/10 h dark). (a) Flowering phenotype. (b) Days at flowering. (c) Dynamic statistics of flowering time. (d) The number of rosette leaves. The seeds of each line were germinated and grown in growth chambers under LD conditions. The days from germination to flowering, the percentage of flowering plants over the same time period, and the total rosette leaf number were measured from 25 plants in each replicate. WT, wild type. bes1-D, untransformed mutant. L9-3 and L9-5 represent complementary lines of ZmBES1/BZR1-9 in the bes1-D mutant. * and ** represent p < 0.05 and p < 0.01, respectively.
Figure 3. The phenotype of complementary lines under LD conditions (14 h light/10 h dark). (a) Flowering phenotype. (b) Days at flowering. (c) Dynamic statistics of flowering time. (d) The number of rosette leaves. The seeds of each line were germinated and grown in growth chambers under LD conditions. The days from germination to flowering, the percentage of flowering plants over the same time period, and the total rosette leaf number were measured from 25 plants in each replicate. WT, wild type. bes1-D, untransformed mutant. L9-3 and L9-5 represent complementary lines of ZmBES1/BZR1-9 in the bes1-D mutant. * and ** represent p < 0.05 and p < 0.01, respectively.
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Plants 12 02995 g004
Figure 4. The phenotype of overexpressed lines under LD conditions (14 h light/10 h dark). (a) Flowering phenotype. (b) Days at flowering. (c) Dynamic statistics of flowering time. (d) The number of rosette leaves. The seeds of each line were germinated and grown in growth chambers under LD conditions. The days from germination to flowering, the percentage of flowering plants over the same time period, and the total rosette leaf number were measured from 25 plants in each replicate. WT, wild type. OE9-2 and OE9-3 represent lines overexpressing ZmBES1/BZR1-9. * and ** represent p < 0.05 and p < 0.01, respectively.
Figure 4. The phenotype of overexpressed lines under LD conditions (14 h light/10 h dark). (a) Flowering phenotype. (b) Days at flowering. (c) Dynamic statistics of flowering time. (d) The number of rosette leaves. The seeds of each line were germinated and grown in growth chambers under LD conditions. The days from germination to flowering, the percentage of flowering plants over the same time period, and the total rosette leaf number were measured from 25 plants in each replicate. WT, wild type. OE9-2 and OE9-3 represent lines overexpressing ZmBES1/BZR1-9. * and ** represent p < 0.05 and p < 0.01, respectively.
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Plants 12 02995 g005
Figure 5. The flowering phenotype of transgenic Arabidopsis under SD conditions. (a) The phenotype of complementary lines. (b) The phenotype of overexpressed lines. (c) The number of rosette leaves. L9-3 and L9-5 represent complementary lines. OE9-2 and OE9-3 represent overexpressing lines. bes1-D, mutant; WT, wild type.
Figure 5. The flowering phenotype of transgenic Arabidopsis under SD conditions. (a) The phenotype of complementary lines. (b) The phenotype of overexpressed lines. (c) The number of rosette leaves. L9-3 and L9-5 represent complementary lines. OE9-2 and OE9-3 represent overexpressing lines. bes1-D, mutant; WT, wild type.
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Plants 12 02995 g006
Figure 6. The relative expression of flowering-related genes in complementary lines (a) and overexpressing lines (b). L9-3 and L9-5 represent complementary lines; OE9-2 and OE9-3 represent overexpressing lines. bes1-D, mutant; WT, wild type. AtACTIN2 was used as a reference. * and ** represent p < 0.05 and p < 0.01, respectively.
Figure 6. The relative expression of flowering-related genes in complementary lines (a) and overexpressing lines (b). L9-3 and L9-5 represent complementary lines; OE9-2 and OE9-3 represent overexpressing lines. bes1-D, mutant; WT, wild type. AtACTIN2 was used as a reference. * and ** represent p < 0.05 and p < 0.01, respectively.
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Plants 12 02995 g007
Figure 7. The flowering phenotype of transgenic rice under SD conditions. (a) Phenotype. (b) Statistics of heading date. The transgenic rice plants were grown in Sanya in winter from mid-Oct to mid-March under natural SD conditions with 9.5–12h light. The values represent means ± SEs from three biological replicates. R9-1 and R9-5 represent transgenic lines. WT, wild type. ** represents p < 0.01.
Figure 7. The flowering phenotype of transgenic rice under SD conditions. (a) Phenotype. (b) Statistics of heading date. The transgenic rice plants were grown in Sanya in winter from mid-Oct to mid-March under natural SD conditions with 9.5–12h light. The values represent means ± SEs from three biological replicates. R9-1 and R9-5 represent transgenic lines. WT, wild type. ** represents p < 0.01.
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Plants 12 02995 g008
Figure 8. DEGs of transgenic rice with ZmBES1/BZR1-9 gene compared with WT. (a) The common DEGs shared by R9−1 and R9−5. (b) GO analysis of common DEGs. (c) The relative expression level of flowering-related genes. R9−1 and R9−5 represent transgenic lines. WT, wild type. * and ** represent p < 0.05 and p < 0.01, respectively.
Figure 8. DEGs of transgenic rice with ZmBES1/BZR1-9 gene compared with WT. (a) The common DEGs shared by R9−1 and R9−5. (b) GO analysis of common DEGs. (c) The relative expression level of flowering-related genes. R9−1 and R9−5 represent transgenic lines. WT, wild type. * and ** represent p < 0.05 and p < 0.01, respectively.
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Liu, Y.; Zhang, H.; Feng, W.; Lin, X.; Gao, A.; Cao, Y.; Yang, Q.; Wang, Y.; Li, W.; Fu, F.; et al. The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice. Plants 2023, 12, 2995. https://doi.org/10.3390/plants12162995

AMA Style

Liu Y, Zhang H, Feng W, Lin X, Gao A, Cao Y, Yang Q, Wang Y, Li W, Fu F, et al. The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice. Plants. 2023; 12(16):2995. https://doi.org/10.3390/plants12162995

Chicago/Turabian Style

Liu, Yuan, Hongwanjun Zhang, Wenqi Feng, Xiaolong Lin, Aijun Gao, Yang Cao, Qingqing Yang, Yingge Wang, Wanchen Li, Fengling Fu, and et al. 2023. "The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice" Plants 12, no. 16: 2995. https://doi.org/10.3390/plants12162995

APA Style

Liu, Y., Zhang, H., Feng, W., Lin, X., Gao, A., Cao, Y., Yang, Q., Wang, Y., Li, W., Fu, F., & Yu, H. (2023). The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice. Plants, 12(16), 2995. https://doi.org/10.3390/plants12162995

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