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. 2a) 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. 2b, 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. 5a). 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. 5b), 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. 6a). 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. 6c, d). LlWOX11 remarkably inhibited transcription from the LlRR9 promoter containing “CGAA” and “CATC” (Fig. 6c), 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.