Table_6_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（AP2/ERF）是植物界独特的调控因子，参与植物生长发育、成熟以及生物和非生物胁迫等整个生命活动过程。在番茄（Solanum lycopersicum）中，存在140个AP2/ERF基因；然而，其功能仍不甚明了。在本研究中，通过比较AP2结构域的第14和第19个氨基酸差异，区分了DREB和ERF亚家族成员。即便在将来自20种植物的68个ERF蛋白的AP2结构域以及番茄DREB和ERF蛋白的基序进行比较后，DREB和ERF蛋白与DRE/CRT及/或GCC框的结合能力仍处于未知状态。在果实发育和成熟过程中，13个DREB和19个ERF亚家族基因的表达呈现出一定的规律性变化，且大多数基因的启动子中存在ARF、DRE/CRT和/或GCC框。这表明，这些基因在果实发育和成熟过程中直接或间接地响应了生长素（IAA）和/或乙烯（ET）信号。此外，其中一些基因可能通过反馈调节IAA或ET的生物合成。此外，番茄中包含16个EAR基序的ERF基因在许多器官中表达，其每百万转录本数（TPM）值在大多数器官中超过其他ERF基因。为了确定包含EAR基序的ERF蛋白中的EAR基序是否具有抑制功能，我们在酵母单杂交（YIH）实验中保留了或删除了它们的EAR基序。结果显示，删除EAR基序后，大多数包含EAR基序的ERF蛋白失去了抑制活性。此外，其中一些在成熟过程中表达。因此，这些包含EAR基序的ERF蛋白通过完成目标基因的DRE/CRT和/或GCC框位点，在平衡其他ERF蛋白的调控功能中发挥着至关重要的作用，确保番茄的正常生长和发育。