Discovery of a cis-regulatory element SaeM involved in dynamic regulation of synergid-specific MYB98

Abstract

MYB98 is a key regulator of the genetic network behind pollen tube attraction toward the female gametophyte. MYB98 is specifically expressed in the synergid cells (SCs), a female gametophyte component cells specialized for pollen tube attraction. However, it had not been clear how exactly MYB98 achieves this specific expression pattern. In the current study, we have determined that a normal SC-specific expression of MYB98 is dependent on a 16-bp-long cis-regulatory element, CATTTACACATTAAAA, freshly named as the “Synergid-specificActivationElement ofMYB98” (SaeM). An 84 bp fragment harboring SaeM in the middle was sufficient to drive exclusively SC-specific expression. The element was present in a significantly large proportion of SC-specific gene promoters and in the promoter of MYB98 homologous genes in the Brassicaceae (pMYB98s). Significance of such family-wide SaeM-like element conservation in exclusive SC-specific expression was confirmed by the Arabidopsis-like activation feature of Brassica oleracea–derived pMYB98 and absence of such feature of pMYB98 derived from a non-Brassicaceae member Prunus persica. Additionally, the yeast-one-hybrid assay showed that the SaeM can be recognized by ANTHOCYANINLESS2 (ANL2) and DAP-seq data further suggested for additional three ANL2 homologs targeting the similar cis-element. Overall, our study has concluded that SaeM plays a crucial role in driving exclusively SC-specific expression of MYB98 and strongly suggests for the involvement of ANL2 and its homologs in its dynamic regulation in planta. Future study on the transcription factors is expected to shed more light on the mechanism behind the process.

Transcription Factors behind MYB98 Regulation: What Does the Discovery of SaeM Suggest?

“…We additionally demonstrated that an 84 bp pMYB98 cis-region harboring SaeM in the middle is sufficient to drive such exclusive expression. We showed that a homeodomain protein, ANL2, and its paralogs HDG1, ETD1, and HDG7 constitute binding potential to SaeM [1]. Studies have shown and suggested that crediting a specific expression pattern of a gene to particular TF might be misleading, as such a pattern could be the result of physical association and/or interaction among additional DNA-binding and non-binding TFs [9][10][11][12].…”

“…Several of the Y1H assay-derived TFs were common across all pMYB98 sub-fragments used in current study (Figures 1b and 2), suggesting direct or indirect association with the shortest fragment. This group includes TFs with REM/B3 (3), bZIP (2), AP2/ERF (2), FHA (2), FAR-related (2), NAC (1), and C2H2 zinc finger (1) domains. Only one TF (GAL1) was common between the TF pools of 60 bp and 139 bp fragments (Figures 1b and 2).…”

“…Hence, we developed a simple workflow for this purpose, considering co-expression and potential physical associations of TFs while predicting their functional roles (Figure 3). In the in vivo reporter expression assay, we observed that the latter region of the functionally active 191 bp pMYB98 region confers a repressive effect [1]. Since this fragment was not used in the Y1H assay, we checked available DNA affinity purification sequencing (DAP-Seq) data for potential TFs binding within this region (i.e., −550 bp to −510 bp).…”

“…MYB98 is pivotal in regulating pollen tube attraction towards the ovule, a critical step in plant pollination and fertilization [1,2]. Synthesis of molecules, particularly cysteine-rich proteins directly involved in pollen tube attraction, depends on exclusive expression of MYB98 at synergid cells (SCs), the accessory but crucial female gametophyte component cells [3].…”

“…Recently, we showed that MYB98 promoter (pMYB98) harbors a cis-element crucially important for its SC-specific expression [1]. Mutation within this 16 bp element, named SaeM (SC-specific activation element of MYB98), essentially led to complete loss of its SC-specific activation.…”