Identification of cis-Acting Elements Recognized by Transcription Factor LlWOX11 in Lilium lancifolium

Identification of cis-Acting Elements Recognized by Transcription Factor LlWOX11 in Lilium lancifolium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of cis-Acting Elements Recognized by Transcription Factor LlWOX11 in Lilium lancifolium Jingyi Bai, Panpan Yang, Mengmeng Bi, Leifeng Xu, Jun Ming This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4354503/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract WOX transcription factors play important roles in plant developmental processes and mainly bind to the WOX-binding element to regulate gene expression. Previously, we characterized a WOX gene from Lilium lancifolium, LlWOX11, positively regulating bulbil formationin, and showed that it bound to the motif of TTAATGAG. However, whether LlWOX11 could bind to other motifs is unclear. In this study, Transcription Factor Centered Yeast One Hybrid (TF-Centered Y1H) was utilized to study the motifs recognized by LlWOX11, and five motifs with seven bases were obtained. In addition to five motifs containing known cis-acting elements: TCAACTC (CAREOSREP1), AGAAAGA (DOFCOREZM/POLLENILELAT52), ACAGTAT (CACTFTPPCA1), we identified that LlWOX11 could bind to two new motifs: TGCGAAA, TCCATCA. We further searched for the core sequences of these motifs by Y1H. Dual-luciferase assay (LUC), Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to further determine that these motifs were bound by LlWOX11 in the plant. In addition, we found that LlWOX11 inhibited the transcription of LlRR9 by binding to the screened motifs in the promoter and promoted bulbil formation. These findings will help to further reveal the functions of WOX protein and the molecular mechanism of bulbil formation regulated by LlWOX11. Lilium Bulbil formation LlWOX11 transcription factor cis-Acting elements Yeast one-Hybrid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message We identified “TCAACTC”, “AGAAAGA”, “ACAGTAT”, “TGCGAAA” and “TCCATCA” which are bound by LlWOX11 through TF-Centered Y1H. LlWOX11 inhibited the transcription of LlRR9 by binding to the screened motifs. Introduction WUSCHEL-related homeobox (WOX) family is a class of transcription factors containing homologous domains, which was divided into 15 families. There are three distinct clades through phylogenetic analyses in the WOX gene family: an ancient clade (WOX10, WOX13, WOX14), an intermediate clade (WOX8, WOX9, WOX11, WOX12), and a modern/WUS clade (WUS, WOX1, WOX2, WOX3, WOX4, WOX5, WOX6, WOX7) (Van der Graaff et al., 2009 ). The characteristic structural feature of WOX family proteins is an N-terminal homeodomain (HD) which consists of 60–66 amino acids (He et al., 2022 ). The HD binds DNA through a helix-turn-helix (HTH) structure. The WOX proteins also contain the specific WUS-box motifs, in the form of T-L-X-L-F-P-X-X (X can be any amino acid) at the C-terminal in the genes in the WUS clade. In addition, some WOX proteins contain an activator domain between the HD and the WUS box consisting of acidic amino acids. WOX family gene transcription factors play a wide variety of roles in plant developmental processes (van der Graaff et al., 2009 ; Willoughby et al., 2021). WOX10, WOX13, and WOX14 participate in root development, callus formation and drought tolerances (Ikeuchi et al., 2022 ; Lv et al., 2023 ). WOX2, WOX3 and WOX8 promoted somatic embryo development in plants (Hassani et al., 2022 ; Long et al., 2023 ; Xu et al., 2023 ). WOX11 and WOX12 play roles in root development and organogenesis (Baesso et al., 2018 ; Xu et al., 2023 ). As a member of the WOX family intermediate clade. PgWOX11 binds to the PgCLE45 promoter to regulate adventitious root branching (Liu et al., 2020 ). A new study shows that OsWOX11 works together with OsSLG2 regulate grain width by affecting the expansion of cells within the spikelet shell in rice (Xiong et al., 2023 ). Liao et al.(2023) found that WOX11 regulates seed dormancy through mediating phytochrome B (PHYB) signaling during the induction stage. WOXs play a role in the regulatory network by binding to the motif in the downstream target gene promoters (Liu et al., 2020 ). Currently, motifs of sequences with a TAAT and a G-Box-like, have been identified (Leibfried et al., 2005 ; Yadav et al., 2011 ; O’Malley et al., 2016 ). QHB, WOX3, and WOX11 proteins also bind to the TTAATGG sequence in rice (Kamiya et al., 2003 ; Zhao et al., 2009 ). WOX11 and WOX12 directly bind to the known WOX-binding cis -elements (TTAATGG) in the promoters of WOX5 and WOX7 to promote root primordia initiation and organogenesis (Hu and Xu, 2016 ). In L.lancifolium , LlWOX11 can bind to “TTAATGAG” in the promoter of LlRR9 to inhibit its transcription, and enhance cytokinin signaling to promote bulbil development (He et al., 2022 ). WUS protein could also specifically recognize the sequence CACGTG (Busch et al., 2010 ), and the appraisal of two binding core sequences for WOX13 (CAAT and TTAA) has been accomplished (Franco-Zorrilla et al., 2014 ). At present, the cis -elements of WOXs reported are mainly WOX-binding elemenst, and more binding elements need to be discovered. Transcription factors (TFs) promote or inhibit downstream gene expression via binding to specific cis -acting elements (Ambrosini et al., 2020 ). Hence, the research on the specific cis -acting element recognized by a TF is of great significance for revealing its function and regulatory mechanisms. At present, the main methods of cis -acting element identification include the Systematic evolution of ligands by exponential enrichment technology (SELEX), chromatin immunoprecipitation (ChIP), Chromatin targeted cleavage and labeling (CUT&Tag) technology (Kaya-Okur et al., 2019 ), Yeast one-Hybrid (Y1H) and Transcription Factor-centered Yeast one Hybrid (TF-centered Y1H). Compared to other technologies, TF-centered Y1H is a new method to analyze the function of specific transcription factors which was developed in 2014 (Ji et al., 2014 ). It was conducted on the basis of Y1H and determined the possible protein and DNA interactions in a short time, which is increasingly being used in plant research due to its advantage of simplicity and efficiency. Previous studies showed that LlWOX11 could promote bulbil formation of L. lancifolium (He et al., 2022 ). Further studies showed that LlWOX11 could regulate directly the transcription of LlRR9 via binding to its promoter containing the sequence of motif “TTAATGAG”(He et al., 2022 ). However, whether LlWOX11 can bind to other motifs remains unknown. In this work, we identified the motifs recognized by LlWOX11 using a TF-Centered Y1H assay (Ji et al., 2014 ) and verified through LUC reporter assay, EMSA and ChIP-PCR. These results suggested that LlWOX11 recognizes more motifs than we previously knew. The identified motifs recognized by LlWOX11 will facilitate to understand the functions of LlWOX11 in-depth and lay a solid foundation for revealing the molecular mechanism of bulbil formation regulated by LlWOX11 in lily. Materials and methods Plants Materials The autotriploid species Lilium lancifolium was used as material in this study. L. lancifolium bulbs of uniform size with 1–2 cm buds were grown in soil in a greenhouse at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China, in September of 2020. Samples of the leaf axil were collected by hand dissection with a double edge blade and quickly freezed with liquid nitrogen for RNA extraction and vector construction. Construction of the bait vector Total RNA was isolated from leaf axils of L. lancifolium according to the instructions of the RNAprep Pure Plant Kit (TIANGEN, Beijing, China). cDNA of leaf axils of L. lancifolium was then obtained by using the TransScript® Uni All-in-One First-Strand cDNA Synthesis SuperMix Kit (Trans, Beijing, China). The coding sequence (CDS) of LlWOX11 was amplified and cloned into vector pGADT7-Rec2 (Clontech, CA, USA) to obtain the bait vector pGADT7-LIWOX11 using the yeast recombination method (Matchmaker™ One-Hybrid Library Construction &Screening Kit). TF-Centered Y1H screening We constructed a random DNA library with 7 base lengths according to Ji et al.(2014). pGADT7-LIWOX11 vector and DNA sequence library were transformed into the Y187 yeast strain together and then were cultivated in the medium of TDO + 3-AT (0, 10, 20, 30, 40, 50, 75, 100 mM). Y1H filter was carried to discover the motifs recognized by LlWOX11 (He et al., 2022 ). The inserts in the positive pHIS2 plasmids were sequenced. The insertion sequences together with the left and the right insertion flanking sequences (“GGG” and “CCC”) were analyzed using the programs of PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and New PLACE ( https://www.dna.affrc.go.jp/PLACE/?action=newplace ) to identify whether they are known cis -acting elements. Core sequence of the screened motif analysis In order to identify the core sequences of the screened motifs, base deletion was performed one by one from the left and right ends of each motif sequence. Serial sequences were generated (Fig. 2 ) and interacted with LlWOX11 to determine the core sequence of screened motif. The sequence inserted into pHIS2 vector was repeated three times. The pHIS2 element library plasmid was co-transformed into Y187 with pGADT7- LlWOX11 vector. Then SD/-Leu/-Trp (DDO) and SD/-His/-Leu/-Trp (TDO) medium supplied with 3-AT (3-Amino-1, 2, 4-triazole) were used to select the transformed yeast cells. Electrophoretic mobility shift assay (EMSA) The CDS of LlWOX11 was incorporated into the MAL-C5X vector (Trans, Beijing, China). The recombinant plasmid was transformed into E. coli strain BL21. The expression of the sequence encoding LlWOX11 fused with the Maltose-binding protein (MBP) was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 3 h. The LlWOX11 protein was isolated and purified according to the instruction manual of the pMAL Protein Fusion &Purification System (NEB, New England, USA). SDS-PAGE was used to examine he purity of the recombinant LlWOX11 protein. The probes labeled with biotin were synthesized by Sangon. The same probe without biotin labeling was used as the competitor. The probes were then incubated with LlWOX11 protein and assayed using a LightShift Chemiluminescent EMSA Kit (Thermo, USA) Chromatin immunoprecipitation (ChIP) assay To confirm whether LlWOX11 protein bind to the screened motifs, ChIP experment was conducted on Nicotiana benthamiana plants expressing 35S::LlWOX11-GFP. The ChIP assay was performed applying EpiQuik™ Plant ChIP Kit (EpigenTek, USA). Firstly, plants were cross-linked with 1% formaldehyde, and chromatin was sheared into fragments with 0.2–0.8 kb length through sonicate. The chromatin fragments was immunoprecipitated with an anti-GFP antibody as a positive control (ChIP+) or a mouse IgG antibody (Beyotime) as a negative control (ChIP-). Immunoprecipitated DNA was purified after reversing the cross-linking. The primers used for the ChIP assay are shown in Table S3. The promoter regions of the cell cycle-related gene (NW_017670315.1) in N. benthamiana was searched for ChIP quantitative PCR, and the promoter fragments containing only the screening motifs were used to detect the binding of LlWOX11 and motifs. The ChIP-qPCR data were analyzed according to the improved approach of Shi et al ( 2014 ). Dual-luciferase (LUC) reporter assay in Nicotiana benthamiana Transient expression analysis in vivo was carried out to further verify the results of Y1H. Four-week-old tobacco plants (Col-0) were used to test the bindings of LlWOX11 and the screened motifs according to the method of He et al ( 2022 ). Three tandem copies of the core sequence of “AACT”, “AAAG”, “AGTA”, “CGAA” and “CATC” were introduced into the pluc-35Rluc vector to drive the LUC reporter gene. The CDs of LlWOX11was inserted into pCAMBIA3301 with the 35S promoter (named as pCAM-LlWOX11) as the effector plasmid using the pEASY-Basic Seamless Cloning and Assembly Kit (Trans, Beijing, China). These reporters and effectors were transformed into Agrobacterium tumefaciens strain EHA105. The empty pCAMBIA3301 was also transformed as a control. Distinct reporters were then coinfiltrated with the effector into N. benthamiana leaves using a syringe. The incubated leaves were sprayed with D-luciferin potassium salt (Beyotime, Shanghai, China), and the fluorescence was detected by a chemiluminescent imaging system (Tanon, Shanghai, China). Searching for DNA motifs in the promoters of genes Previous studies showed that LlWOX11 binds to the promoter of LlRR9 and activates its expression. In addition, we found a positive correlation between the expression levels of some genes concerned with plant development and LlWOX11 in L. lancifolium . In order to comprehend the distribution of screened motifs, 1000bp promoter sequence before “ATG” of these genes from Arabidopsis thaliana were retrieved from the NCBI database ( https://www.ncbi.nlm.nih.gov/ ) for analysis. Statistical analysis The data for analyses were presented as the means with standard deviation from three biological replicates. SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses, and statistical significance was defined at p < 0.05. Non-overlapping letters indicated significant differences between the comparisons, based on the ANOVA analysis and Duncan’s multiple range test. Results Construction and screening of the random DNA insertion prey library Through plate screening with different concentrations, yeast cells cannot grow on the medium plate containing 75 mM 3AT (Fig. 1 a), indicating the reporter gene cannot be activated. According to the results, 75 mM 3AT was selected for subsequent screening concentrations. The Y187 yeast strain containing pGADT7-LIWOX11 bait plasmid was used as the receptor strain. The motif library plasmid was transferred into it and coated on an SD-TLH screening plate containing 75 mM 3AT. Screening through the TDO medium with 75 mM 3-AT, 155 positive clones were selected (Fig. 1 b). Identification of the DNA motifs bound by LlWOX11 The TF-Centered Y1H system is a convenient and efficient approach to identify the DNA sequence bound by transcription factors of interest. TF-centered Y1H was performed, and five positive clones that have high binding affinity to LlWOX11 were identified. Through high stringency selection, the sequences of the eleven insert fragments are obtained (Table 1 ). Motif prediction showed that three of the sequences contained known cisacting elements: CAREOSREP1, DOFCOREZM/POLLENILELAT52 and CACTFTPPCA1 (Table 1 ). No studied element was predicted in the other insertion sequences: “TGCGAAA” and “TCCATCA”, and thus these sequences might contain novel motifs recognized by LlWOX11. Table 1 Analysis of the insertion sequences possibly bound by LlWOX11. Clone number The identified sequence (underlined) with the two sides of flanking sequences (5’-3’) Motif prediction 1 CCCTCAACTCGGG CAREOSREP1 2 CCCAGAAAGAGGG DOFCOREZM; POLLENILELAT52 3 CCCACAGTATGGG CACTFTPPCA1 4 CCCTGCGAAAGGG No result 5 CCCTCCATCAGGG No result Core sequence characterization of the motifs recognized by LlWOX11 We used Y1H assays to identify the core sequence of the filtered motifs. Yeast cells containing the reporter vector with the three repeated tandem motifs having the third DNA base (A) missing from the left border of studied motif CAREOSREP1 (TCAACTC) could not grow on TDO medium with 75 mmol/L 3-AT, illustrating that LlWOX11 bound to the sequence “AACTC”, but not to “ACTC”. Moreover, cells with the reporter lacking the second DNA base (T) from the right border did not grow, implying that LlWOX11 is able to bind to the sequence “AACT”, but not to“AAC” (Fig. 2 ). These observations indicated that the third DNA base (A) from the left border and the second DNA base (T) from the right border of motif CAREOSREP1 are critical for binding by LlWOX11. In the same way, the unannotated motifs were determined (Fig. 2 ). The core sequences of the remaining two annotated motifs are “AAAG”, “AGTA”. The core sequences of two unknown motifs are “CGAA”, “CATC” respectively. EMSA analysis of the bindings of the selected motifs with LlWOX11 To further determine whether these motifs were truly bound by LlWOX11, EMSA assays were subsequently performed. The EMSA results of five motif probes revealed retarded band, which demonstrates that DNA–protein complexes could be observed when LlWOX11 interacted with these five motifs, and the signal decreased when the same unlabeled competitor probe was added (Fig. 3 a), showing that LlWOX11 could bind to these selected motifs, which was consistent with the results of Y1H. The core sequence of the screened motif was also subjected to EMSA analysis, and the results showed that the core sequence binds to the LlWOX11 protein (Fig. 3 b), consistent with the Y1H results. Interaction of LlWOX11 with selected motifs actually occurs in plants We performed ChIP analysis to determine whether bindings between LlWOX11 and the screened motifs actually occur in plants or not. The LlWOX11-GFP fusion gene was transformed into tobacco ( N. benthamiana ) plants. The N. benthamiana genes (NW_017670315.1), which contained the screened motifs in promoter regions, were used in this experiment (Fig. 4 a). The promoter fragments containing specific motifs were captured by ChIP using a GFP antibody. Then, the enriched truncated promoter identified by ChIP-PCR was utilized for ChIP-qPCR, and both the promoter fragments containing the selected motifs were significantly enriched (Fig. 4 b, c). Meanwhile, the promoter fragment (P1) lacking any sequences of the selected motifs was used as negative controls, and it failed to be enriched. The results suggested that the bindings between LlWOX11 and the screened motifs to regulate gene expression actually occurred in plants. The distribution of screened motifs in the promoters of genes regulated by LlWOX11 According to the transcriptome sequencing data, LlWOX11 could induce the expression of 61 genes significantly. These genes involved “signal transduction mechanisms”, “cytokinin metabolic process” and “cell cycle” based on Cluster of Orthologous Groups of proteins (COG) analysis, and included two transcription factor families, NAC and MYB. Analyzing the distribution of “AACT”, “AAAG”, “AGTA”, “CGAA” and “CATC” in the promoters (− 1 to − 1000 bp) of these genes in A. thaliana , and the results show that all of the promoters of the genes contained the screened motif respectively (Fig. 5 a). We speculate that LlWOX11 further regulates the expression of these genes by binding to the screened motifs in the promoters. The frequency of the five motifs occurs in the promoters of different cluster genes is basically consistent, with “AAAG” having the highest frequency of occurrence, followed by “AACT”, and the frequency of occurrence of “CGAA” being the smallest (Fig. 5 b). DOFCOREZM/POLLENILELAT52 motifs were abundant and the large number of “AAAG” may be related to the wide range of functions of this element. LlWOX11 inhibits the transcription of LlRR9 by binding to the screened motifs in the promoter Previous studies have shown that LlWOX11 can bind to the promoter of LlRR9 (He et al., 2022 ). After promoter sequence analysis, 4-class selected motifs were distributed on the promoter of LlRR9 (Fig. 6 a). These core sequences of the 4-class motif were fused with the LUC gene to construct reporter vectors (Fig. 6 b), and each reporter vector was co-transformed with the effector into tobacco plants. The results showed that LlWOX11 could bind to all the studied core sequences (Fig. 6 c). To determine whether LlWOX11 could bind to selected motifs to regulate gene expression, truncated promoters only containing one core sequence of the selected motifs were selected for study. These truncated promoter fragments were used as probes for EMSA, and the DNA-protein complexes generated when the LlWOX11 protein bind to the labeled probe, and more same unlabeled probe, the lower signal of the bound complex will be. When the LlWOX11 protein interacted with the mutant probes, no signal was detectable (Fig. 6 d). These results indicated that LlWOX11 could bind to the core sequence“AACT”, “AAAG”, “CGAA”, “CATC”. This result was consistent with that of the LUC assay, which further confirmed that LlWOX11 could regulate gene expression by binding to the core sequence “AACT”, “AAAG”, “CGAA”, “CATC”. Discussion TFs are central regulators of changes in gene expression and have fundamental importance in critical aspects of plant growth and development. TFs regulate target gene expression by binding to specific cis-acting elements (Ambrosini et al., 2020 ), and studying the element bound by a TF is important to display its regulatory mechanism. TF-centered Y1H is a new method to analyze the function of specific TFs which is increasingly being used in plant research. This is a useful method to research protein-DNA interactions in Tamarix hispida (Xu et al., 2017 ), Betula hygrometrica (Guo et al., 2018 ), Populus simonii × Populus nigra ) (Wang et al., 2021 ) and rice (Chen et al., 2022 ), but there is no report on lily. Previous studies showed that LlWOX11 is positively correlated with bulbil formation of L. lancifolium (He et al., 2022 ), but its downstream regulatory mechanism is unclear. In the present study, we applied this method in Lily for the first time to investigate the downstream motifs bound by LlWOX11. Five motifs (three known, two unknown) were obtained (Table 1 ) and laid a solid foundation for displaying the regulation mechanism on molecular level of LlWOX11 in bulbil formation. WOX TFs are plant-specific transcription factors, several studies showed specifically sequences with a TAAT and a G-Box like have been identified as bound by WOXs (Leibfried et al., 2005 ; Yadav et al., 2011 ; O’ Malley et al., 2016), and more components remain to be mined. In our result, five motifs containing three known cis-acting elements which were annotated separately as CAREOSREP1 (TCAACTC), DOFCOREZM/POLLENILELAT52 (AGAAAGA), CACTFTPPCA1 (ACAGTAT). Several CAREs involved in plant growth and development were identified in carrot, poplar and other plant species (Liu et al., 2019 ; Campos et al, 2021 ). BpCUC2 directly binds to CAREOSREP1 which was found extensively on the promoter in a series of IAA-related and cyclin-related genes in Betula pendula . BpCUC2 was speculated to participating in normal internode and leaf development by regulating the expression of IAA-related and cyclin-related genes (Liu et al., 2019 ), which provides a reference for us to search for downstream genes of LlWOX11. Gibberellin treatment promotes bulbil formation in yam (Kim et al., 2003 ), gibberellin (GA)-responsive element CAREOSREP1 was found in our screening result (Table 1 ), from which we speculate that LlWOX11 might play a role in the gibberellin pathway by binding to “TCAACTC”. MdWOX11 promotes the formation of adventitious root(AR) primordia via binding to the WOX binding site (WOX-box) element CCATTAA, TTAATGG and ACC(A/T)(A/C/T)(A/C/T)” in apple (Mao et al., 2023 ). In our study, LlWOX11 protein could bind the sequence “TCAACTC”, in which the core sequence of “AACT” (Fig. 2 a) are similar with WOX-box. It indicated that the conserved sequence “AA(C/T)” may be significant for the binding of WOXs. TFs bind to definite motifs and act in regulating gene expression, so their binding motifs could be used to predict their function. The DOFCOREZM/POLLENILELAT52 motif was found previously to be bound by CDF transcription factors, which have a dof domain identifying the DOFCOREZM/ POLLENILELAT52 motif (Imaizumi et al., 2005 ). CDFs regulate the expression of MYB60, SP5G by binding to the DOFCOREZM/POLLENILELAT52 motif in its promoters, and are involved in regulating potato tuberization and enhancement of activity in guard cells (Cominelli et al., 2011 ; Lehretz et al., 2019 ; Zhang et al., 2023 ). CDF transcription factors play important roles in plant growth and development (Jin et al., 2024 ). Therefore, the binding of LlWOX11 to the DOFCOREZM/POLLENILELAT52 motif (Fig. 2 b, 3 , 4 ) suggested that it might have similar functions to the CDF TFs, such as being mediated by sucrose signals (Qi et al., 2020 ), and it is consistent with previous research (Hao et al., 2023), which further demonstrated that LlWOX11 is involved in the regulation of bulbil formation by responding to sucrose signaling. “TGCGAAA” and “TCCATCA” are novel motifs identified to be bound by WOX11, which have not been reported. Our results showed that they are significantly inhibited by LlWOX11 and widely present in the promoter regions of transcription factors involved in plant growth and development, which suggested that the “TGCGAAA” and “TCCATCA” elements are also significant motifs recognized by WOXs, and should receive further study. The sequence of a cis-acting element exhibits differences in individual bases across different species (Lohmann et al., 2001 ; Mao et al., 2023 ). Screening of the core sequence of screening elements can find the working sequence information, which contributes to the identification and analysis of elements. We further screened the core sequences “AACT”, “AAAG”, “AGTA”, “CGAA”, “CATC” of both annotated and new motifs by deleting bases separately (Fig. 2 ). The binding of LlWOX11 to the screened motifs was genuine through EMSA (Fig. 3 ). The resulting sequence information has important implications for the study of the LlWOX11-binding elements. Within the organism, the number and positional distribution of motifs within the promoter region are associated with the transcription machinery due to the folding of the DNA in the three-dimensional structure (Casimiro et al., 2008 ). Our analysis found that the screening motifs were distributed on the promoters of genes involved in “signal transduction mechanisms”, “cytokinin metabolic process” and “cell cycle” in Arabidopsis (Fig. 5 a). The bulbil in L. lancifolium originated from the leaf axillary meristem, and the formation process was associated with cell division and regulated by cytokinin (He et al., 2022 ). Therefore, these genes may be the key genes involved in the pearl bud formation regulated by LlWOX11. The distribution patterns are diverse and may be different in relation their functions in bulbil formation. The high number of annotated motifs may be related to the wide function of these motifs in plants, while the new motifs are a small number (Fig. 5 b), we speculate that it has a specific function. Transcription factors can function in combination with multiple elements (Guo et al., 2018 ; Wang et al., 2021 ). LlRR9 is a type-A RR gene whose product is a negative regulator of cytokinin signaling. Our previous studies have found that LlRR9 participated in bulbil formation and was directly regulated by LlWOX11 through the WOX-binding element (TTAATGAG) in its promoter (He et al., 2022 ). In this study, we found sequences containing the core sequence of four classes of screened motifs (“AACT”, “AAAG”, “CGAA”, “CATC”) in the promoter of LlRR9 (Fig. 6 a). We carried out dual-luciferase reporter and EMSAs using promoter fragments containing only the screening motifs, and the results showed that LlWOX11 affect transcription from the LlRR9 promoter by binding to the screened motifs (Fig. 6 c, d). LlWOX11 remarkably inhibited transcription from the LlRR9 promoter containing “CGAA” and “CATC” (Fig. 6 c), which is consistent with previous research. The fluorescence signal of “AACT” and “AAAG” has not decreased, which may be related to the extensive functions of “AAAG” and the complex regulatory network of LlWOX11 in regulating bulbil formation. In conclusion, we construct a prey library and combine it with high-throughput sequencing to completely determine the motif in lily. We identified “TCAACTC”, “AGAAAGA”, “ACAGTAT”, “TGCGAAA” and “TCCATCA”, which are recognized by LlWOX11, and they probably play significant part in LlWOX11-mediated gene expression. The identification of the five motifs will lay the foundation for the in-depth characterization of the functions of WOX11 and provide a reference for further research on the molecular regulatory pathway the formation of bulbils in L. lancifolium . Declarations Conflict of interest The authors declare that they have no conflict of interest in relation to this work. Declarations Conflict of interest The authors declare that they have no relevant financial or non-financial interests to disclose. Funding This work was supported by National Natural Science Foundation of China (32172612, 31902043), National key R& D program of China (2019YFD1001002) . Acknowledgment We very much appreciate suggestions on the experimental operation by Dr. Guoren He (Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University). We appreciate researcher Tiegang Lu and his team (Biotechnology Research Institute of Chinese Academy of Agricultural Sciences) for their theoretical suggestions. We appreciate Ruiyuan Biotechnology (Nanjing) for its technical support during the process of TF-centered Y1H. References Ambrosini G, Vorontsov I, Penzar D, Groux R, Fornes O, Nikolaeva DD, Ballester B, Grau J, Grosse I, Makeev V, Kulakovskiy I, Bucher P (2020) Insights gained from a comprehensive all-against-all transcription factor binding motif benchmarking study. 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Plant Biotechnol J 19:2139–2379 Wang YM, Zhang YM, Zhang X, Zhao X, Zhang Y, Wang C, Wang YC, Wang LQ (2021) Poplar PsnICE1 enhances cold tolerance by binding to different cis-acting elements to improve reactive oxygen species-scavenging capability. Tree Physiol 41(12):2424–2437. 10.1093/treephys/tpab084 Willoughby AC, Nimchuk Z (2021) WOX going on: CLE peptides in plant development. Curr Opin Plant Biol 63:102056. 10.1016/j.pbi.2021.102056 Xiong DP, Wang R, Wang YM, Li Y, Sun G, Yao SG (2023) SLG2 specifically regulates grain width through WOX11 -mediated cell expansion control in rice. Plant Biotechnol J 21:1904–1918. 10.1111/pbi.14102 Xu AJ, Yang JQ, Wang SQ, Zheng L, Wang J, Zhang YW, Bi XJ, Wang H (2023) Characterization and expression profiles of WUSCHEL-related homeobox (WOX) gene family in cultivated alfalfa ( Medicago sativa L). 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Plant Cell 21:736–748 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4354503","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309091879,"identity":"033b72d8-93a4-4130-b952-dfad70eedda6","order_by":0,"name":"Jingyi Bai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACA2YGhgMMDDb8YF5CAfFa0iQbwFoMiNECoQ5DtDAQo8Wcnffh4YJf5yX4JbITPzwwYJDnFzuAX4tlM7vB4Zl9tyUke85ulgA6zHDm7AQCDjvMxnCYt+d2ncHx3g0gLQkGt4nTck7C4DDv5h/Ea+H5cUACaMs24myxbAbZ0pAM8ss2iwQDCcJ+Mec/xvyZ548dMMRyN9/8UWEjzy9NQAsYMLbBmRJEKAeDP8QqHAWjYBSMghEJAEIeQIt0XLKTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6445-9492","institution":"Southwest Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Bai","suffix":""},{"id":309091880,"identity":"04446c99-c224-4feb-859f-fb12611b7f8a","order_by":1,"name":"Panpan Yang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Yang","suffix":""},{"id":309091881,"identity":"2eb83eff-58b0-4462-936c-26584ac4921a","order_by":2,"name":"Mengmeng Bi","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Bi","suffix":""},{"id":309091882,"identity":"3012eb56-d610-41a0-a2ac-bd96a24d301d","order_by":3,"name":"Leifeng Xu","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Leifeng","middleName":"","lastName":"Xu","suffix":""},{"id":309091883,"identity":"8a48319c-0ca1-431e-a7f2-af92fca5f30f","order_by":4,"name":"Jun Ming","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ming","suffix":""}],"badges":[],"createdAt":"2024-05-01 13:27:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4354503/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4354503/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58386426,"identity":"1645d8e7-2485-4cb3-958d-50b6c358dbbc","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1645735,"visible":true,"origin":"","legend":"\u003cp\u003eThe motifs combined with LlWOX11 were screened out by TF-centered Y1H Assay. (a) Detection of pGADT7-LlWOX11 Self-Activation in Y187 Yeast. (b) Verification of positive clones in TF-centered Y1H assay.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/e6ce0c9c9231fb05ae46642c.png"},{"id":58386432,"identity":"569a6062-05f0-468b-8ad0-f3a066ca9438","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":597731,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the core sequence of the screened motifs recognized by LlWOX11. The serial sequences (D1–D6) were interacted with LlWOX11 using Y1H to determine the core motif. p53HIS2/pGADT7-p53 and p53HIS2/pGADT7-LlWOX11 were used as the positive control and negative control, respectively. The underlined bases represent the screened 5′ and 3′ ends of the core region.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/f97b7ab184e9bf30d68209b7.png"},{"id":58386428,"identity":"e3a0e74a-7e05-4456-9307-ca7a03eef43f","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273874,"visible":true,"origin":"","legend":"\u003cp\u003eEMSA analysis of the bindings of the selected motifs with LlWOX11. (a) EMSA analysis of the bindings of the selected motifs with LlWOX11. The five selected motifs (motifs 1-5) were used for EMSA analysis. The competitor for the labeled probe was tested by adding a 200-fold excess of the same unlabeled probe. Arrows indicate the DNA-protein complexes and free probes. (b) EMSA analysis of the bindings of LlWOX11 with the core sequence of the five motifs. The tandem repeated core sequences of five screened motifs were used for EMSA analysis. The competitor for the labeled probe was tested by adding a 200-fold excess of the same unlabeled probe. Arrows indicate the DNA-protein complexes and free probes.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/eccfecba9da7d293c136a110.png"},{"id":58386429,"identity":"d5b1b2bb-3fce-4c77-97e6-c7654c7b7295","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":314440,"visible":true,"origin":"","legend":"\u003cp\u003eChIP assay analysis of the binding of LlWOX11 to selected motifs in plants. (a) A schematic diagram of the location of promoter regions containing the screened motifs used for ChIP, the gray rectangular frame indicates the promoter regions containing screened motifs used for ChIP. (b) ChIP-PCR detection of LlWOX11. (c) The promoter fragments containing one of the screened motifs were amplified for ChIP-qPCR. An asterisk indicates a significant difference between IgG and Anti-GFP at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/3cef91c7399ffcf67bb932c0.png"},{"id":58386431,"identity":"72857388-4425-4f74-9940-9b5d75920e92","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":511006,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of screened motifs in the promoters of genes probably regulated by LlWOX11 in \u003cem\u003eA. thaliana\u003c/em\u003e. (a) The distribution and number of screened motifs in the promoters of genes related to“signal transduction mechanisms”, “cytokinin metabolic process”, “cell cycle” \u003cem\u003eNACs\u003c/em\u003e and \u003cem\u003eMYBs\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e. (b) The proportion of individual motifs among the five motifs in the promoters of different clustered genes.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/f6394dbecb9f6fc9a2d94c67.png"},{"id":58386430,"identity":"0015cb60-f742-4ab5-8312-2e1e0845d0c5","added_by":"auto","created_at":"2024-06-14 18:44:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":339359,"visible":true,"origin":"","legend":"\u003cp\u003eThe binding of LlWOX11 to the core sequence of screened motifs in promoter of \u003cem\u003eLlRR9\u003c/em\u003e. (a) The distribution of four screened motifs in the promoter of \u003cem\u003eLlRR9\u003c/em\u003e (-1 to -1000bp). (b, c) Diagrams of the reporter and effector vectors are shown. Three tandem copies of the core sequence of four screened motifs were inserted into pluc-35Rluc and were co-transformed with 35S: LlWOX11 into tobacco. (d) EMSA assay of the binding of LlWOX11 to the four screened motifs. EMSA was performed using the purified LlWOX11 protein and the four screened motifs or the mutant as probes. 50, 200-fold excess of the unlabeled probes were added to test the Competition for the same labeled probe. The mutated probe was used as the negative control.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/08d431cab31a523c724719ae.png"},{"id":59605068,"identity":"16be040c-51c1-48ba-b0b7-9054d73870ed","added_by":"auto","created_at":"2024-07-03 18:29:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5207498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4354503/v1/eab8c951-12fb-4b2c-a180-ec0002f9f8da.pdf"}],"financialInterests":"","formattedTitle":"Identification of cis-Acting Elements Recognized by Transcription Factor LlWOX11 in Lilium lancifolium","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cstrong\u003eWe identified \u0026ldquo;TCAACTC\u0026rdquo;, \u0026ldquo;AGAAAGA\u0026rdquo;, \u0026ldquo;ACAGTAT\u0026rdquo;, \u0026ldquo;TGCGAAA\u0026rdquo; and \u0026ldquo;TCCATCA\u0026rdquo; which are bound by LlWOX11 through TF-Centered Y1H. LlWOX11 inhibited the transcription of \u003cem\u003eLlRR9\u003c/em\u003e by binding to the screened motifs.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eWUSCHEL-related homeobox (WOX) family is a class of transcription factors containing homologous domains, which was divided into 15 families. There are three distinct clades through phylogenetic analyses in the WOX gene family: an ancient clade (WOX10, WOX13, WOX14), an intermediate clade (WOX8, WOX9, WOX11, WOX12), and a modern/WUS clade (WUS, WOX1, WOX2, WOX3, WOX4, WOX5, WOX6, WOX7) (Van der Graaff et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The characteristic structural feature of WOX family proteins is an N-terminal homeodomain (HD) which consists of 60\u0026ndash;66 amino acids (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The HD binds DNA through a helix-turn-helix (HTH) structure. The WOX proteins also contain the specific WUS-box motifs, in the form of T-L-X-L-F-P-X-X (X can be any amino acid) at the C-terminal in the genes in the WUS clade. In addition, some WOX proteins contain an activator domain between the HD and the WUS box consisting of acidic amino acids.\u003c/p\u003e \u003cp\u003eWOX family gene transcription factors play a wide variety of roles in plant developmental processes (van der Graaff et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Willoughby et al., 2021). WOX10, WOX13, and WOX14 participate in root development, callus formation and drought tolerances (Ikeuchi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lv et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). WOX2, WOX3 and WOX8 promoted somatic embryo development in plants (Hassani et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Long et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). WOX11 and WOX12 play roles in root development and organogenesis (Baesso et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a member of the WOX family intermediate clade. PgWOX11 binds to the \u003cem\u003ePgCLE45\u003c/em\u003e promoter to regulate adventitious root branching (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A new study shows that OsWOX11 works together with OsSLG2 regulate grain width by affecting the expansion of cells within the spikelet shell in rice (Xiong et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Liao et al.(2023) found that WOX11 regulates seed dormancy through mediating phytochrome B (PHYB) signaling during the induction stage. WOXs play a role in the regulatory network by binding to the motif in the downstream target gene promoters (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, motifs of sequences with a TAAT and a G-Box-like, have been identified (Leibfried et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; O\u0026rsquo;Malley et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). QHB, WOX3, and WOX11 proteins also bind to the TTAATGG sequence in rice (Kamiya et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). WOX11 and WOX12 directly bind to the known WOX-binding \u003cem\u003ecis\u003c/em\u003e-elements (TTAATGG) in the promoters of \u003cem\u003eWOX5\u003c/em\u003e and \u003cem\u003eWOX7\u003c/em\u003e to promote root primordia initiation and organogenesis (Hu and Xu, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In \u003cem\u003eL.lancifolium\u003c/em\u003e, LlWOX11 can bind to \u0026ldquo;TTAATGAG\u0026rdquo; in the promoter of \u003cem\u003eLlRR9\u003c/em\u003e to inhibit its transcription, and enhance cytokinin signaling to promote bulbil development (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). WUS protein could also specifically recognize the sequence CACGTG (Busch et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and the appraisal of two binding core sequences for WOX13 (CAAT and TTAA) has been accomplished (Franco-Zorrilla et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). At present, the \u003cem\u003ecis\u003c/em\u003e-elements of WOXs reported are mainly WOX-binding elemenst, and more binding elements need to be discovered.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) promote or inhibit downstream gene expression via binding to specific \u003cem\u003ecis\u003c/em\u003e-acting elements (Ambrosini et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hence, the research on the specific \u003cem\u003ecis\u003c/em\u003e-acting element recognized by a TF is of great significance for revealing its function and regulatory mechanisms. At present, the main methods of \u003cem\u003ecis\u003c/em\u003e-acting element identification include the Systematic evolution of ligands by exponential enrichment technology (SELEX), chromatin immunoprecipitation (ChIP), Chromatin targeted cleavage and labeling (CUT\u0026amp;Tag) technology (Kaya-Okur et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Yeast one-Hybrid (Y1H) and Transcription Factor-centered Yeast one Hybrid (TF-centered Y1H). Compared to other technologies, TF-centered Y1H is a new method to analyze the function of specific transcription factors which was developed in 2014 (Ji et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It was conducted on the basis of Y1H and determined the possible protein and DNA interactions in a short time, which is increasingly being used in plant research due to its advantage of simplicity and efficiency.\u003c/p\u003e \u003cp\u003ePrevious studies showed that \u003cem\u003eLlWOX11\u003c/em\u003e could promote bulbil formation of \u003cem\u003eL. lancifolium\u003c/em\u003e (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further studies showed that \u003cem\u003eLlWOX11\u003c/em\u003e could regulate directly the transcription of \u003cem\u003eLlRR9\u003c/em\u003e via binding to its promoter containing the sequence of motif \u0026ldquo;TTAATGAG\u0026rdquo;(He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, whether LlWOX11 can bind to other motifs remains unknown. In this work, we identified the motifs recognized by LlWOX11 using a TF-Centered Y1H assay (Ji et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and verified through LUC reporter assay, EMSA and ChIP-PCR. These results suggested that LlWOX11 recognizes more motifs than we previously knew. The identified motifs recognized by LlWOX11 will facilitate to understand the functions of LlWOX11 in-depth and lay a solid foundation for revealing the molecular mechanism of bulbil formation regulated by LlWOX11 in lily.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlants Materials\u003c/h2\u003e \u003cp\u003eThe autotriploid species \u003cem\u003eLilium lancifolium\u003c/em\u003e was used as material in this study. \u003cem\u003eL. lancifolium\u003c/em\u003e bulbs of uniform size with 1\u0026ndash;2 cm buds were grown in soil in a greenhouse at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China, in September of 2020. Samples of the leaf axil were collected by hand dissection with a double edge blade and quickly freezed with liquid nitrogen for RNA extraction and vector construction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the bait vector\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from leaf axils of \u003cem\u003eL. lancifolium\u003c/em\u003e according to the instructions of the RNAprep Pure Plant Kit (TIANGEN, Beijing, China). cDNA of leaf axils of \u003cem\u003eL. lancifolium\u003c/em\u003e was then obtained by using the TransScript\u0026reg; Uni All-in-One First-Strand cDNA Synthesis SuperMix Kit (Trans, Beijing, China). The coding sequence (CDS) of \u003cem\u003eLlWOX11\u003c/em\u003e was amplified and cloned into vector pGADT7-Rec2 (Clontech, CA, USA) to obtain the bait vector pGADT7-LIWOX11 using the yeast recombination method (Matchmaker\u0026trade; One-Hybrid Library Construction \u0026amp;Screening Kit).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTF-Centered Y1H screening\u003c/h2\u003e \u003cp\u003eWe constructed a random DNA library with 7 base lengths according to Ji et al.(2014). pGADT7-LIWOX11 vector and DNA sequence library were transformed into the Y187 yeast strain together and then were cultivated in the medium of TDO\u0026thinsp;+\u0026thinsp;3-AT (0, 10, 20, 30, 40, 50, 75, 100 mM). Y1H filter was carried to discover the motifs recognized by LlWOX11 (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The inserts in the positive pHIS2 plasmids were sequenced. The insertion sequences together with the left and the right insertion flanking sequences (\u0026ldquo;GGG\u0026rdquo; and \u0026ldquo;CCC\u0026rdquo;) were analyzed using the programs of PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and New PLACE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dna.affrc.go.jp/PLACE/?action=newplace\u003c/span\u003e\u003cspan address=\"https://www.dna.affrc.go.jp/PLACE/?action=newplace\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify whether they are known \u003cem\u003ecis\u003c/em\u003e-acting elements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCore sequence of the screened motif analysis\u003c/h2\u003e \u003cp\u003eIn order to identify the core sequences of the screened motifs, base deletion was performed one by one from the left and right ends of each motif sequence. Serial sequences were generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and interacted with LlWOX11 to determine the core sequence of screened motif. The sequence inserted into pHIS2 vector was repeated three times. The pHIS2 element library plasmid was co-transformed into Y187 with pGADT7- LlWOX11 vector. Then SD/-Leu/-Trp (DDO) and SD/-His/-Leu/-Trp (TDO) medium supplied with 3-AT (3-Amino-1, 2, 4-triazole) were used to select the transformed yeast cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eThe CDS of \u003cem\u003eLlWOX11\u003c/em\u003e was incorporated into the MAL-C5X vector (Trans, Beijing, China). The recombinant plasmid was transformed into E. coli strain BL21. The expression of the sequence encoding LlWOX11 fused with the Maltose-binding protein (MBP) was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37\u0026deg;C for 3 h. The LlWOX11 protein was isolated and purified according to the instruction manual of the pMAL Protein Fusion \u0026amp;Purification System (NEB, New England, USA). SDS-PAGE was used to examine he purity of the recombinant LlWOX11 protein. The probes labeled with biotin were synthesized by Sangon. The same probe without biotin labeling was used as the competitor. The probes were then incubated with LlWOX11 protein and assayed using a LightShift Chemiluminescent EMSA Kit (Thermo, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h2\u003e \u003cp\u003eTo confirm whether LlWOX11 protein bind to the screened motifs, ChIP experment was conducted on \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants expressing 35S::LlWOX11-GFP. The ChIP assay was performed applying EpiQuik\u0026trade; Plant ChIP Kit (EpigenTek, USA). Firstly, plants were cross-linked with 1% formaldehyde, and chromatin was sheared into fragments with 0.2\u0026ndash;0.8 kb length through sonicate. The chromatin fragments was immunoprecipitated with an anti-GFP antibody as a positive control (ChIP+) or a mouse IgG antibody (Beyotime) as a negative control (ChIP-). Immunoprecipitated DNA was purified after reversing the cross-linking. The primers used for the ChIP assay are shown in Table S3. The promoter regions of the cell cycle-related gene (NW_017670315.1) in \u003cem\u003eN. benthamiana\u003c/em\u003e was searched for ChIP quantitative PCR, and the promoter fragments containing only the screening motifs were used to detect the binding of LlWOX11 and motifs. The ChIP-qPCR data were analyzed according to the improved approach of Shi et al (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual-luciferase (LUC) reporter assay in\u003c/b\u003e \u003cb\u003eNicotiana benthamiana\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTransient expression analysis in vivo was carried out to further verify the results of Y1H. Four-week-old tobacco plants (Col-0) were used to test the bindings of LlWOX11 and the screened motifs according to the method of He et al (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Three tandem copies of the core sequence of \u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;AGTA\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo; and \u0026ldquo;CATC\u0026rdquo; were introduced into the pluc-35Rluc vector to drive the LUC reporter gene. The CDs of LlWOX11was inserted into pCAMBIA3301 with the 35S promoter (named as pCAM-LlWOX11) as the effector plasmid using the pEASY-Basic Seamless Cloning and Assembly Kit (Trans, Beijing, China). These reporters and effectors were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105. The empty pCAMBIA3301 was also transformed as a control. Distinct reporters were then coinfiltrated with the effector into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using a syringe. The incubated leaves were sprayed with D-luciferin potassium salt (Beyotime, Shanghai, China), and the fluorescence was detected by a chemiluminescent imaging system (Tanon, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSearching for DNA motifs in the promoters of genes\u003c/h2\u003e \u003cp\u003ePrevious studies showed that LlWOX11 binds to the promoter of \u003cem\u003eLlRR9\u003c/em\u003e and activates its expression. In addition, we found a positive correlation between the expression levels of some genes concerned with plant development and LlWOX11 in \u003cem\u003eL. lancifolium\u003c/em\u003e. In order to comprehend the distribution of screened motifs, 1000bp promoter sequence before \u0026ldquo;ATG\u0026rdquo; of these genes from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were retrieved from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data for analyses were presented as the means with standard deviation from three biological replicates. SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses, and statistical significance was defined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Non-overlapping letters indicated significant differences between the comparisons, based on the ANOVA analysis and Duncan\u0026rsquo;s multiple range test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and screening of the random DNA insertion prey library\u003c/h2\u003e \u003cp\u003eThrough plate screening with different concentrations, yeast cells cannot grow on the medium plate containing 75 mM 3AT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), indicating the reporter gene cannot be activated. According to the results, 75 mM 3AT was selected for subsequent screening concentrations. The Y187 yeast strain containing pGADT7-LIWOX11 bait plasmid was used as the receptor strain. The motif library plasmid was transferred into it and coated on an SD-TLH screening plate containing 75 mM 3AT. Screening through the TDO medium with 75 mM 3-AT, 155 positive clones were selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the DNA motifs bound by LlWOX11\u003c/h2\u003e \u003cp\u003eThe TF-Centered Y1H system is a convenient and efficient approach to identify the DNA sequence bound by transcription factors of interest. TF-centered Y1H was performed, and five positive clones that have high binding affinity to LlWOX11 were identified. Through high stringency selection, the sequences of the eleven insert fragments are obtained (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Motif prediction showed that three of the sequences contained known cisacting elements: CAREOSREP1, DOFCOREZM/POLLENILELAT52 and CACTFTPPCA1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No studied element was predicted in the other insertion sequences: \u0026ldquo;TGCGAAA\u0026rdquo; and \u0026ldquo;TCCATCA\u0026rdquo;, and thus these sequences might contain novel motifs recognized by LlWOX11.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of the insertion sequences possibly bound by LlWOX11.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClone number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThe identified sequence (underlined)\u003c/p\u003e \u003cp\u003ewith the two sides of flanking sequences (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMotif prediction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCTCAACTCGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAREOSREP1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCAGAAAGAGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDOFCOREZM; POLLENILELAT52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCACAGTATGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACTFTPPCA1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCTGCGAAAGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo result\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCTCCATCAGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo result\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCore sequence characterization of the motifs recognized by LlWOX11\u003c/h2\u003e \u003cp\u003eWe used Y1H assays to identify the core sequence of the filtered motifs. Yeast cells containing the reporter vector with the three repeated tandem motifs having the third DNA base (A) missing from the left border of studied motif CAREOSREP1 (TCAACTC) could not grow on TDO medium with 75 mmol/L 3-AT, illustrating that LlWOX11 bound to the sequence \u0026ldquo;AACTC\u0026rdquo;, but not to \u0026ldquo;ACTC\u0026rdquo;. Moreover, cells with the reporter lacking the second DNA base (T) from the right border did not grow, implying that LlWOX11 is able to bind to the sequence \u0026ldquo;AACT\u0026rdquo;, but not to\u0026ldquo;AAC\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These observations indicated that the third DNA base (A) from the left border and the second DNA base (T) from the right border of motif CAREOSREP1 are critical for binding by LlWOX11. In the same way, the unannotated motifs were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The core sequences of the remaining two annotated motifs are \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;AGTA\u0026rdquo;. The core sequences of two unknown motifs are \u0026ldquo;CGAA\u0026rdquo;, \u0026ldquo;CATC\u0026rdquo; respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEMSA analysis of the bindings of the selected motifs with LlWOX11\u003c/h2\u003e \u003cp\u003eTo further determine whether these motifs were truly bound by LlWOX11, EMSA assays were subsequently performed. The EMSA results of five motif probes revealed retarded band, which demonstrates that DNA\u0026ndash;protein complexes could be observed when LlWOX11 interacted with these five motifs, and the signal decreased when the same unlabeled competitor probe was added (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), showing that LlWOX11 could bind to these selected motifs, which was consistent with the results of Y1H. The core sequence of the screened motif was also subjected to EMSA analysis, and the results showed that the core sequence binds to the LlWOX11 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), consistent with the Y1H results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInteraction of LlWOX11 with selected motifs actually occurs in plants\u003c/h2\u003e \u003cp\u003eWe performed ChIP analysis to determine whether bindings between LlWOX11 and the screened motifs actually occur in plants or not. The LlWOX11-GFP fusion gene was transformed into tobacco (\u003cem\u003eN. benthamiana\u003c/em\u003e) plants. The \u003cem\u003eN. benthamiana\u003c/em\u003e genes (NW_017670315.1), which contained the screened motifs in promoter regions, were used in this experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The promoter fragments containing specific motifs were captured by ChIP using a GFP antibody. Then, the enriched truncated promoter identified by ChIP-PCR was utilized for ChIP-qPCR, and both the promoter fragments containing the selected motifs were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Meanwhile, the promoter fragment (P1) lacking any sequences of the selected motifs was used as negative controls, and it failed to be enriched. The results suggested that the bindings between LlWOX11 and the screened motifs to regulate gene expression actually occurred in plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe distribution of screened motifs in the promoters of genes regulated by LlWOX11\u003c/h2\u003e \u003cp\u003eAccording to the transcriptome sequencing data, LlWOX11 could induce the expression of 61 genes significantly. These genes involved \u0026ldquo;signal transduction mechanisms\u0026rdquo;, \u0026ldquo;cytokinin metabolic process\u0026rdquo; and \u0026ldquo;cell cycle\u0026rdquo; based on Cluster of Orthologous Groups of proteins (COG) analysis, and included two transcription factor families, NAC and MYB. Analyzing the distribution of \u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;AGTA\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo; and \u0026ldquo;CATC\u0026rdquo; in the promoters (\u0026minus;\u0026thinsp;1 to \u0026minus;\u0026thinsp;1000 bp) of these genes in \u003cem\u003eA. thaliana\u003c/em\u003e, and the results show that all of the promoters of the genes contained the screened motif respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We speculate that LlWOX11 further regulates the expression of these genes by binding to the screened motifs in the promoters. The frequency of the five motifs occurs in the promoters of different cluster genes is basically consistent, with \u0026ldquo;AAAG\u0026rdquo; having the highest frequency of occurrence, followed by \u0026ldquo;AACT\u0026rdquo;, and the frequency of occurrence of \u0026ldquo;CGAA\u0026rdquo; being the smallest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). DOFCOREZM/POLLENILELAT52 motifs were abundant and the large number of \u0026ldquo;AAAG\u0026rdquo; may be related to the wide range of functions of this element.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLlWOX11 inhibits the transcription of\u003c/b\u003e \u003cb\u003eLlRR9\u003c/b\u003e \u003cb\u003eby binding to the screened motifs in the promoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies have shown that LlWOX11 can bind to the promoter of \u003cem\u003eLlRR9\u003c/em\u003e (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After promoter sequence analysis, 4-class selected motifs were distributed on the promoter of \u003cem\u003eLlRR9\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). These core sequences of the 4-class motif were fused with the LUC gene to construct reporter vectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), and each reporter vector was co-transformed with the effector into tobacco plants. The results showed that LlWOX11 could bind to all the studied core sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). To determine whether LlWOX11 could bind to selected motifs to regulate gene expression, truncated promoters only containing one core sequence of the selected motifs were selected for study. These truncated promoter fragments were used as probes for EMSA, and the DNA-protein complexes generated when the LlWOX11 protein bind to the labeled probe, and more same unlabeled probe, the lower signal of the bound complex will be. When the LlWOX11 protein interacted with the mutant probes, no signal was detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). These results indicated that LlWOX11 could bind to the core sequence\u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo;, \u0026ldquo;CATC\u0026rdquo;. This result was consistent with that of the LUC assay, which further confirmed that LlWOX11 could regulate gene expression by binding to the core sequence \u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo;, \u0026ldquo;CATC\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTFs are central regulators of changes in gene expression and have fundamental importance in critical aspects of plant growth and development. TFs regulate target gene expression by binding to specific cis-acting elements (Ambrosini et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and studying the element bound by a TF is important to display its regulatory mechanism. TF-centered Y1H is a new method to analyze the function of specific TFs which is increasingly being used in plant research. This is a useful method to research protein-DNA interactions in \u003cem\u003eTamarix hispida\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eBetula hygrometrica\u003c/em\u003e (Guo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003ePopulus simonii\u003c/em\u003e\u0026times;\u003cem\u003ePopulus nigra\u003c/em\u003e) (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and rice (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but there is no report on lily. Previous studies showed that LlWOX11 is positively correlated with bulbil formation of \u003cem\u003eL. lancifolium\u003c/em\u003e (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but its downstream regulatory mechanism is unclear. In the present study, we applied this method in Lily for the first time to investigate the downstream motifs bound by LlWOX11. Five motifs (three known, two unknown) were obtained (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and laid a solid foundation for displaying the regulation mechanism on molecular level of LlWOX11 in bulbil formation.\u003c/p\u003e \u003cp\u003eWOX TFs are plant-specific transcription factors, several studies showed specifically sequences with a TAAT and a G-Box like have been identified as bound by WOXs (Leibfried et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; O\u0026rsquo; Malley et al., 2016), and more components remain to be mined. In our result, five motifs containing three known cis-acting elements which were annotated separately as CAREOSREP1 (TCAACTC), DOFCOREZM/POLLENILELAT52 (AGAAAGA), CACTFTPPCA1 (ACAGTAT). Several CAREs involved in plant growth and development were identified in carrot, poplar and other plant species (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Campos et al, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BpCUC2 directly binds to CAREOSREP1 which was found extensively on the promoter in a series of IAA-related and cyclin-related genes in \u003cem\u003eBetula pendula\u003c/em\u003e. BpCUC2 was speculated to participating in normal internode and leaf development by regulating the expression of IAA-related and cyclin-related genes (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which provides a reference for us to search for downstream genes of LlWOX11. Gibberellin treatment promotes bulbil formation in yam (Kim et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), gibberellin (GA)-responsive element CAREOSREP1 was found in our screening result (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), from which we speculate that LlWOX11 might play a role in the gibberellin pathway by binding to \u0026ldquo;TCAACTC\u0026rdquo;. \u003cem\u003eMdWOX11\u003c/em\u003e promotes the formation of adventitious root(AR) primordia via binding to the WOX binding site (WOX-box) element CCATTAA, TTAATGG and ACC(A/T)(A/C/T)(A/C/T)\u0026rdquo; in apple (Mao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our study, LlWOX11 protein could bind the sequence \u0026ldquo;TCAACTC\u0026rdquo;, in which the core sequence of \u0026ldquo;AACT\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) are similar with WOX-box. It indicated that the conserved sequence \u0026ldquo;AA(C/T)\u0026rdquo; may be significant for the binding of WOXs. TFs bind to definite motifs and act in regulating gene expression, so their binding motifs could be used to predict their function. The DOFCOREZM/POLLENILELAT52 motif was found previously to be bound by CDF transcription factors, which have a dof domain identifying the DOFCOREZM/ POLLENILELAT52 motif (Imaizumi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). CDFs regulate the expression of MYB60, SP5G by binding to the DOFCOREZM/POLLENILELAT52 motif in its promoters, and are involved in regulating potato tuberization and enhancement of activity in guard cells (Cominelli et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lehretz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). CDF transcription factors play important roles in plant growth and development (Jin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, the binding of LlWOX11 to the DOFCOREZM/POLLENILELAT52 motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) suggested that it might have similar functions to the CDF TFs, such as being mediated by sucrose signals (Qi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and it is consistent with previous research (Hao et al., 2023), which further demonstrated that LlWOX11 is involved in the regulation of bulbil formation by responding to sucrose signaling. \u0026ldquo;TGCGAAA\u0026rdquo; and \u0026ldquo;TCCATCA\u0026rdquo; are novel motifs identified to be bound by WOX11, which have not been reported. Our results showed that they are significantly inhibited by LlWOX11 and widely present in the promoter regions of transcription factors involved in plant growth and development, which suggested that the \u0026ldquo;TGCGAAA\u0026rdquo; and \u0026ldquo;TCCATCA\u0026rdquo; elements are also significant motifs recognized by WOXs, and should receive further study.\u003c/p\u003e \u003cp\u003eThe sequence of a cis-acting element exhibits differences in individual bases across different species (Lohmann et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Mao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Screening of the core sequence of screening elements can find the working sequence information, which contributes to the identification and analysis of elements. We further screened the core sequences \u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;AGTA\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo;, \u0026ldquo;CATC\u0026rdquo; of both annotated and new motifs by deleting bases separately (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The binding of LlWOX11 to the screened motifs was genuine through EMSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The resulting sequence information has important implications for the study of the LlWOX11-binding elements. Within the organism, the number and positional distribution of motifs within the promoter region are associated with the transcription machinery due to the folding of the DNA in the three-dimensional structure (Casimiro et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Our analysis found that the screening motifs were distributed on the promoters of genes involved in \u0026ldquo;signal transduction mechanisms\u0026rdquo;, \u0026ldquo;cytokinin metabolic process\u0026rdquo; and \u0026ldquo;cell cycle\u0026rdquo; in Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The bulbil in \u003cem\u003eL. lancifolium\u003c/em\u003e originated from the leaf axillary meristem, and the formation process was associated with cell division and regulated by cytokinin (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, these genes may be the key genes involved in the pearl bud formation regulated by LlWOX11. The distribution patterns are diverse and may be different in relation their functions in bulbil formation. The high number of annotated motifs may be related to the wide function of these motifs in plants, while the new motifs are a small number (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), we speculate that it has a specific function.\u003c/p\u003e \u003cp\u003eTranscription factors can function in combination with multiple elements (Guo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eLlRR9\u003c/em\u003e is a type-A RR gene whose product is a negative regulator of cytokinin signaling. Our previous studies have found that LlRR9 participated in bulbil formation and was directly regulated by LlWOX11 through the WOX-binding element (TTAATGAG) in its promoter (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we found sequences containing the core sequence of four classes of screened motifs (\u0026ldquo;AACT\u0026rdquo;, \u0026ldquo;AAAG\u0026rdquo;, \u0026ldquo;CGAA\u0026rdquo;, \u0026ldquo;CATC\u0026rdquo;) in the promoter of LlRR9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We carried out dual-luciferase reporter and EMSAs using promoter fragments containing only the screening motifs, and the results showed that LlWOX11 affect transcription from the LlRR9 promoter by binding to the screened motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). LlWOX11 remarkably inhibited transcription from the \u003cem\u003eLlRR9\u003c/em\u003e promoter containing \u0026ldquo;CGAA\u0026rdquo; and \u0026ldquo;CATC\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), which is consistent with previous research. The fluorescence signal of \u0026ldquo;AACT\u0026rdquo; and \u0026ldquo;AAAG\u0026rdquo; has not decreased, which may be related to the extensive functions of \u0026ldquo;AAAG\u0026rdquo; and the complex regulatory network of LlWOX11 in regulating bulbil formation.\u003c/p\u003e \u003cp\u003eIn conclusion, we construct a prey library and combine it with high-throughput sequencing to completely determine the motif in lily. We identified \u0026ldquo;TCAACTC\u0026rdquo;, \u0026ldquo;AGAAAGA\u0026rdquo;, \u0026ldquo;ACAGTAT\u0026rdquo;, \u0026ldquo;TGCGAAA\u0026rdquo; and \u0026ldquo;TCCATCA\u0026rdquo;, which are recognized by LlWOX11, and they probably play significant part in LlWOX11-mediated gene expression. The identification of the five motifs will lay the foundation for the in-depth characterization of the functions of WOX11 and provide a reference for further research on the molecular regulatory pathway the formation of bulbils in \u003cem\u003eL. lancifolium\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest in relation to this work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (32172612, 31902043), National key R\u0026amp; D program of China (2019YFD1001002) .\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe very much appreciate suggestions on the experimental operation by Dr. Guoren He (Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University). We appreciate researcher Tiegang Lu and his team (Biotechnology Research Institute of Chinese Academy of Agricultural Sciences) for their theoretical suggestions. We appreciate Ruiyuan Biotechnology (Nanjing) for its technical support during the process of TF-centered Y1H.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmbrosini G, Vorontsov I, Penzar D, Groux R, Fornes O, Nikolaeva DD, Ballester B, Grau J, Grosse I, Makeev V, Kulakovskiy I, Bucher P (2020) Insights gained from a comprehensive all-against-all transcription factor binding motif benchmarking study. 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Plant Cell 21:736\u0026ndash;748\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lilium, Bulbil formation, LlWOX11 transcription factor, cis-Acting elements, Yeast one-Hybrid","lastPublishedDoi":"10.21203/rs.3.rs-4354503/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4354503/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWOX transcription factors play important roles in plant developmental processes\u0026nbsp;and mainly bind to the WOX-binding element to regulate gene expression. Previously, we characterized a WOX gene from Lilium lancifolium, LlWOX11, positively regulating bulbil formationin, and showed that it bound to the motif of TTAATGAG. However, whether LlWOX11 could bind to other motifs is unclear. In this study, Transcription Factor Centered Yeast One Hybrid (TF-Centered Y1H) was utilized to study the motifs recognized by LlWOX11, and five motifs with seven bases were obtained. In addition to five motifs containing known cis-acting elements: TCAACTC\u0026nbsp;(CAREOSREP1), AGAAAGA\u0026nbsp;(DOFCOREZM/POLLENILELAT52), ACAGTAT\u0026nbsp;(CACTFTPPCA1), we identified that LlWOX11 could bind to two new motifs: TGCGAAA, TCCATCA. We further searched for the core sequences of these motifs by Y1H. Dual-luciferase assay (LUC), Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to further determine that these motifs were bound by LlWOX11 in the plant. In addition, we found that LlWOX11 inhibited the transcription of LlRR9\u0026nbsp;by binding to the screened motifs in the promoter and promoted bulbil formation. These findings will help to further reveal the functions of WOX protein and the molecular mechanism of bulbil formation regulated by LlWOX11.\u003c/p\u003e","manuscriptTitle":"Identification of cis-Acting Elements Recognized by Transcription Factor LlWOX11 in Lilium lancifolium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 18:44:36","doi":"10.21203/rs.3.rs-4354503/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfa3b083-00e1-4aa5-82a7-4e0d27be5ab6","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-03T18:21:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-14 18:44:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4354503","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4354503","identity":"rs-4354503","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}