Lee Jae-Hoon, Hong Jong-Pil, Oh Sang-Keun, Lee Sanghyeob, Choi Doil, Kim Woo Taek
Department of Biology, College of Science, Yonsei University, South Korea.
Plant Mol Biol. 2004 May;55(1):61-81. doi: 10.1007/s11103-004-0417-6.
From a pathogen-inoculated hot pepper (Capsicum annuum L. cv. Pukang) leaf EST, we identified a cDNA clone, pCaERFLP1, encoding a putative transcription factor that contains a single ERF/AP2 DNA binding domain. CaERFLP1 was most closely related to tomato LeERF2 (73%), both of which belong to the novel ERF class IV typified by the N-terminal MCGGAIL signature sequence, while it had a limited sequence identity (25-30%) with Arabidopsis AtERFs and tobacco NtERFs. Quantitative gel retardation assays revealed that bacterially expressed full-length CaERFLP1 was able to form a specific complex with both the GCC box and DRE/CRT motif, with its binding affinity for GCC being stronger than for DRE/CRT. When fused to the GAL4 DNA binding domain, the N-terminal CaERFLP1(1-37) and C-terminal CaERFLP1(198-264) mutant polypeptides could function individually as transactivators in yeast. This suggests that two separate domains of CaERFLP1 may play distinct roles in transcription activation. In particle co-bombardment experiments, CaERFLP1 activated the transcription of reporter genes containing the 4X[GCC] element in tobacco cells. In hot pepper plants, the steady-state level of CaERFLP1 mRNA was markedly induced by multiple environmental factors, such as pathogen infection, ethylene, mechanical wounding and high salinity. Furthermore, ectopic expression of CaERFLP1 in transgenic tobacco plants resulted in partially improved tolerance against the bacterial pathogen Pseudomonas syringae and salt stress (100 mM NaCl). Consistently, various defense-related genes, including GCC box-containing PR genes and the DRE/CRT-containing LTI45 (ERD10) gene, were constitutively expressed in 35S::CaERFLP1 tobacco plants. Thus, it appears that CaERFLP1 is functional in tobacco cells, where it induces the transactivation of some GCC- and DRE/CRT-genes to trigger a subset of stress response. Here, the possible biological role(s) of CaERFLP1 is discussed, especially with regard to the possibility that CaERFLP1 has multiple functions in the regulation of GCC- and DRE/CRT-mediated gene expression in hot pepper plants.
从接种病原体的辣椒（Capsicum annuum L. cv. Pukang）叶片EST中，我们鉴定出一个cDNA克隆pCaERFLP1，其编码一个推定的转录因子，该转录因子含有单个ERF/AP2 DNA结合结构域。CaERFLP1与番茄LeERF2的亲缘关系最为密切（73%），二者均属于以N端MCGGAIL特征序列为代表的新型IV类ERF，而它与拟南芥AtERFs和烟草NtERFs的序列同一性有限（25 - 30%）。定量凝胶阻滞分析表明，细菌表达的全长CaERFLP1能够与GCC盒和DRE/CRT基序形成特异性复合物，其对GCC的结合亲和力强于对DRE/CRT的结合亲和力。当与GAL4 DNA结合结构域融合时，N端CaERFLP1(1 - 37)和C端CaERFLP1(198 - 264)突变多肽在酵母中可单独作为反式激活因子发挥作用。这表明CaERFLP1的两个独立结构域可能在转录激活中发挥不同作用。在粒子共轰击实验中，CaERFLP1激活了烟草细胞中含有4X[GCC]元件的报告基因的转录。在辣椒植株中，CaERFLP1 mRNA的稳态水平受到多种环境因子的显著诱导，如病原体感染、乙烯、机械损伤和高盐度。此外，CaERFLP1在转基因烟草植株中的异位表达导致对细菌病原体丁香假单胞菌和盐胁迫（100 mM NaCl）的耐受性部分提高。一致地，包括含GCC盒的PR基因和含DRE/CRT的LTI45（ERD10）基因在内的多种防御相关基因在35S::CaERFLP1烟草植株中组成型表达。因此，CaERFLP1似乎在烟草细胞中发挥功能，在其中它诱导一些GCC和DRE/CRT基因的反式激活以触发一部分应激反应。在此，讨论了CaERFLP1可能的生物学作用，特别是关于CaERFLP1在辣椒植株中调控GCC和DRE/CRT介导的基因表达方面具有多种功能的可能性。
