WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiflorum ‘white heaven’). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca2+. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was significantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca2+-CaM and multiprotein bridging factor pathways.

中文翻译：

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LlWRKY39 通过激活 LlMBF1c 并与百合 (Lilium longiflorum) 中的 LlCaM3 相互作用参与耐热性

WRKY 转录因子 (TFs) 在植物对不同非生物胁迫的反应中非常重要。然而，关于它们在耐热性调节中的作用的研究仍然有限。在这里，我们研究了 LlWRKY39 在百合耐热性中的作用（百合'白色天堂'）。根据多重比对分析，LlWRKY39 属于 WRKY IId 亚类，包含一个潜在的钙调蛋白 (CaM) 结合域。进一步的分析表明 LlCaM3 通过与其 CaM 结合域结合与 LlWRKY39 相互作用，这种相互作用取决于 Ca2+.LlWRKY39由热应激（HS）诱导，在细胞核中检测到LlWRKY39-GFP融合蛋白。百合和拟南芥的耐热性随着异位过表达而增加LlWRKY39. 热相关基因的表达HSFA1,HSFA2,AtMBF1c,AtGolS1,在DREB2A,在WRKY39， 和AtHSP101在转基因拟南芥品系中显着升高，这可能促进了耐热性的增加。那么，发起人LlMBF1c从百合中分离，发现 LlWRKY39 与其启动子中的保守 W-box 元件结合以激活其活性，表明 LlWRKY39 是LlMBF1c. 此外，双荧光素酶报告基因分析表明，通过蛋白质相互作用，LlCaM3 对 LlWRKY39 的转录激活产生负面影响。LlMBF1c，这可能是平衡LlWRKY39介导的热应激反应（HSR）的重要反馈调节途径。总的来说，这些结果意味着 LlWRKY39 可能通过 Ca 参与 HSR 作为重要的调节因子。2+-CaM 和多蛋白桥接因子途径。