Table_5_Function Analysis of the ERF and DREB Subfamilies in Tomato Fruit Development and Ripening.DOCX

APETALA2/ethylene responsive factors (AP2/ERF) are unique regulators in the plant kingdom and are involved in the whole life activity processes such as development, ripening, and biotic and abiotic stresses. In tomato (Solanum lycopersicum), there are 140 AP2/ERF genes; however, their functionality remains poorly understood. In this work, the 14th and 19th amino acid differences in the AP2 domain were used to distinguish DREB and ERF subfamily members. Even when the AP2 domain of 68 ERF proteins from 20 plant species and motifs in tomato DREB and ERF proteins were compared, the binding ability of DREB and ERF proteins with DRE/CRT and/or GCC boxes remained unknown. During fruit development and ripening, the expressions of 13 DREB and 19 ERF subfamily genes showed some regular changes, and the promoters of most genes had ARF, DRE/CRT, and/or GCC boxes. This suggests that these genes directly or indirectly respond to IAA and/or ethylene (ET) signals during fruit development and ripening. Moreover, some of these may feedback regulate IAA or ET biosynthesis. In addition, 16 EAR motif-containing ERF genes in tomato were expressed in many organs and their total transcripts per million (TPM) values exceeded those of other ERF genes in most organs. To determine whether the EAR motif in EAR motif-containing ERF proteins has repression function, their EAR motifs were retained or deleted in a yeast one-hybrid (YIH) assay. The results indicate that most of EAR motif-containing ERF proteins lost repression activity after deleting the EAR motif. Moreover, some of these were expressed during ripening. Thus, these EAR motif-containing ERF proteins play vital roles in balancing the regulatory functions of other ERF proteins by completing the DRE/CRT and/or GCC box sites of target genes to ensure normal growth and development in tomato.

APETALA2/乙烯响应因子（APETALA2/ethylene responsive factors，AP2/ERF）是植物界特有的调控因子，参与植物生长发育、果实成熟以及生物与非生物胁迫响应等全部生命活动进程。在番茄（Solanum lycopersicum）中，共存在140个AP2/ERF家族基因，但其功能仍未得到充分阐释。本研究通过AP2结构域（AP2 domain）中第14位与第19位氨基酸的差异，区分DREB与ERF亚家族成员。即便对20个植物物种的68个ERF蛋白的AP2结构域，以及番茄DREB和ERF蛋白的基序进行比对后，DREB与ERF蛋白结合DRE/CRT和/或GCC盒的能力仍未明确。在果实发育与成熟过程中，13个DREB亚家族基因与19个ERF亚家族基因的表达呈现规律性变化，且多数基因的启动子区域含有ARF、DRE/CRT以及/或GCC盒元件。这表明上述基因在果实发育与成熟阶段，可直接或间接响应吲哚乙酸（Indole-3-acetic acid，IAA）以及/或乙烯（Ethylene，ET）信号；此外，其中部分基因可能通过反馈调控IAA或ET的生物合成。此外，番茄中16个含有EAR基序（EAR motif）的ERF基因在多个器官中均有表达，且其每百万转录本（Transcripts Per Million，TPM）值在多数器官中均高于其他ERF基因。为明确含EAR基序的ERF蛋白中的EAR基序是否具备转录抑制功能，本研究通过酵母单杂交（Yeast One-Hybrid，YIH）实验，对这类蛋白的EAR基序进行了保留或敲除处理。结果显示，多数含EAR基序的ERF蛋白在敲除EAR基序后丧失了转录抑制活性；且其中部分基因在果实成熟阶段存在表达。综上，这类含EAR基序的ERF蛋白可通过填补靶基因的DRE/CRT和/或GCC盒结合位点，平衡其他ERF蛋白的调控功能，从而保障番茄的正常生长与发育。