Department of Bioenergy Science and Technology, Chonnam National University, Buk-Gu, Gwangju, 61186, South Korea.
Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Buk-Gu, Gwangju, 61186, South Korea.
Planta. 2023 Jun 24;258(2):26. doi: 10.1007/s00425-023-04183-3.
LBD18 and IAA14 antagonistically interact with ARF7 through the electrostatic faces in the ARF7PB1 domain, modulating ARF7 transcriptional activity. Auxin Response Factor 7 (ARF7)/ARF19 control lateral root development by directly activating Lateral Organ Boundaries Domain 16 (LBD16)/LBD18 genes in Arabidopsis. LBD18 upregulates ARF19 expression by binding to the ARF19 promoter. It also interacts with ARF7 through the Phox and Bem1 (PB1) domain to enhance the ARF7 transcriptional activity, forming a dual mode of positive feedback loop. LBD18 competes with the repressor indole-3-acetic acid 14 (IAA14) for ARF7 binding through the PB1 domain. In this study, we examined the molecular determinant of the ARF7 PB1 domain for interacting with LBD18 and showed that the electronic faces in the ARF7 PB1 domain are critical for interacting with LBD18 and IAA14/17. We used a luminescence complementation imaging assay to determine protein-protein interactions. The results showed that mutation of the invariant lysine residue and the OPCA motif in the PB1 domain in ARF7 significantly reduces the protein interaction between ARF7 and LBD18. Transient gene expression assays with Arabidopsis protoplasts showed that IAA14 suppressed transcription-enhancing activity of LBD18 on the LUC reporter gene fused to the ARF19 promoter harboring an auxin response element, but mutation of the invariant lysine residue and OPCA motif in the PB1 domain of IAA14 reduced the repression capability of IAA14 for transcription-enhancing activity of LBD18. We further showed that the same mutation in the PB1 domain of IAA14 reduces its repression capability, thereby increasing the LUC activity induced by both ARF7 and LBD18 compared with IAA14. These results suggest that LBD18 competes with IAA14 for ARF7 binding via the electrostatic faces of the ARF7 PB1 domain to modulate ARF7 transcriptional activity.
LBD18 和 IAA14 通过 ARF7PB1 结构域中的静电面拮抗相互作用，调节 ARF7 的转录活性。在拟南芥中，生长素响应因子 7(ARF7)/ARF19 通过直接激活侧根边界域 16(LBD16)/LBD18 基因来控制侧根的发育。LBD18 通过与 ARF19 启动子结合而上调 ARF19 的表达。它还通过 Phox 和 Bem1(PB1)结构域与 ARF7 相互作用，增强 ARF7 的转录活性，形成双重正反馈回路模式。LBD18 通过 PB1 结构域与抑制物吲哚-3-乙酸 14(IAA14)竞争 ARF7 的结合。在这项研究中，我们研究了 ARF7 PB1 结构域与 LBD18 相互作用的分子决定因素，并表明 ARF7 PB1 结构域中的电子面对于与 LBD18 和 IAA14/17 的相互作用至关重要。我们使用发光互补成像测定法来确定蛋白质-蛋白质相互作用。结果表明，ARF7 PB1 结构域中不变的赖氨酸残基和 OPCA 基序的突变显著降低了 ARF7 与 LBD18 之间的蛋白质相互作用。用拟南芥原生质体进行瞬时基因表达试验表明，IAA14 抑制了 LBD18 在含有生长素反应元件的 ARF19 启动子融合的 LUC 报告基因上的转录增强活性，但 IAA14 的 PB1 结构域中不变的赖氨酸残基和 OPCA 基序的突变降低了 IAA14 对 LBD18 转录增强活性的抑制能力。我们进一步表明，IAA14 的 PB1 结构域中的相同突变降低了其抑制能力，从而增加了 ARF7 和 LBD18 诱导的 LUC 活性，与 IAA14 相比。这些结果表明，LBD18 通过 ARF7 PB1 结构域的静电面与 IAA14 竞争 ARF7 的结合，从而调节 ARF7 的转录活性。
