College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An 271018, China.
Plant Physiol. 2024 Mar 29;194(4):2472-2490. doi: 10.1093/plphys/kiae017.
LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.
侧向器官边界域/不对称叶 2 样（LBD/ASLs）是植物特有的转录因子，它们在生长素调控的侧根（LR）形成的下游起作用。我们之前的研究发现 PpLBD16 正向调控桃（Prunus persica）LR 的形成。然而，PpLBD16 的下游调控网络和靶基因在很大程度上仍然未知。在这里，我们构建了 PpLBD16 同源过表达系和 PpLBD16 沉默系。我们发现过表达 PpLBD16 促进了桃根的起始，而沉默 PpLBD16 抑制了桃根的形成。通过 PpLBD16 过表达和沉默系根的 RNA 测序（RNA-seq）分析，我们发现 PpLBD16 正向调控的基因与细胞壁合成和降解、离子/物质转运以及离子结合和稳态密切相关。为了进一步检测 PpLBD16 的结合基序和潜在靶基因，我们进行了体外 DNA 亲和纯化测序（DAP-seq）分析。PpLBD16 优先结合 CCNGAAANNNNGG（MEME-1）、[C/T]TTCT[C/T][T/C]（MEME-2）和 GCGGCGG（ABR1）基序。通过 RNA-seq 和 DAP-seq 数据的综合分析，我们筛选了 PpLBD16 的候选靶基因。我们证明 PpLBD16 结合并激活细胞壁修饰相关基因 EXPANSIN-B2（PpEXPB2）和 SUBTILISIN-LIKE PROTEASE 1.7（PpSBT1.7）、离子转运相关基因 CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1（PpCNGC1）和多酚氧化酶（PPO）编码基因 PpPPO，从而控制桃根器官发生并促进 LR 的形成。此外，我们的结果显示 PpLBD16 及其靶基因参与了桃 LR 原基的发育。总的来说，这项工作揭示了 PpLBD16 的下游调控网络和靶基因，为 LBD16 介导的 LR 发育的分子网络提供了新的见解。
