Function of AP2/ERF transcription factors in phytochrome signalling

为了使生长和发育适应环境，植物不断地利用专门的光感受器监测周围的光条件。光敏色素是一种对红光和远红光敏感的感光器。感光后，光敏色素在细胞核中积累，并调节数百个基因的表达，这些基因控制种子萌发、幼苗建立、开花诱导和生长发育的许多其他方面。种子萌发和休眠是受光和赤霉素(GA)和脱落酸(ABA)控制的重要生态和农艺性状。ERF55和ERF58是两个密切相关的AP2/ERF转录因子，我们发现它们是在种子中表达的新的光敏色素相互作用蛋白，我们发现在erf55 erf58突变体中对光依赖的种子萌发有促进作用。光敏色素结合ERF55/ERF58，取代PIF1、SOM、ABI5等萌发抑制基因的启动子，以及编码ABA合成代谢酶或GA分解代谢酶的基因，从而促进萌发。根据初步数据，我们假设ERF55和ERF58也在决定休眠深度方面发挥作用，以响应发育中的种子所感知的光环境。在拟议项目的第一个工作包中，我们旨在验证这一假说，并使用DAP-seq来鉴定具有调节休眠功能的ERF55和ERF58的靶基因。ERF55和ERF58属于AP2/ERF转录因子家族的A6亚组。这个亚组的所有成员都与光敏色素相互作用，但--除了ERF55和ERF58--在幼苗或成年植物中表达，而不是在种子中表达。因此，在第二个工作包中，我们旨在利用CRISPR/Cas9产生的ERF高位突变体和DAP-seq高通量筛选潜在的靶基因来鉴定这些AP2/ERF在幼苗和成年植物光信号转导中的功能。光敏色素与AP2/ERF高度保守的DNA结合域结合，表明光敏色素与AP2/ERF的结合可能在AP2/ERF中广泛存在。从不同亚群中随机挑选的几个AP2/ERF确实与光敏色素相互作用。因此，在第三个工作包中，我们希望筛选整个AP2/ERF家族，寻找与光敏色素相互作用的成员。综上所述，我们已经确定AP2/ERF转录家族成员是光敏色素的新的相互作用因子。在拟议的项目中，我们希望研究它们在光敏色素介导的光反应中的功能，并阐明其潜在的分子机制。

In order to adapt growth and development to the environment, plants constantly monitor the light conditions in their surrounding using dedicated photoreceptors. Phytochromes are photoreceptors sensitive to the red and far-red range of the light spectrum. Upon sensing light, phytochromes accumulate in the nucleus and regulate the expression of hundreds of genes that control seed germination, seedling establishment, induction of flowering, and many other aspects of growth and development. Seed germination and seed dormancy are important ecological and agronomic traits controlled by light and the phythormones abscisic acid (ABA) and gibberellin (GA). ERF55 and ERF58, two closely related AP2/ERF transcription factors that we identified as novel phytochrome interacting proteins, are expressed in seeds and we found that light-dependent seed germination is enhanced in the erf55 erf58 mutant. Light-activated phytochromes bind ERF55/ERF58 and displace them from promoters of germination-repressing genes such as PIF1, SOM, ABI5, and genes coding for ABA anabolic or GA catabolic enzymes, thereby promoting germination. Based on preliminary data, we hypothesise that ERF55 and ERF58 also play a role in setting the depth of dormancy in response to the light environment perceived by the developing seeds. In the first work package of the proposed project, we aim to test this hypothesis and use DAP-seq to identify target genes of ERF55 and ERF58 with a function in regulation of dormancy. ERF55 and ERF58 belong to subgroup A6 of the AP2/ERF transcription factor family. All members of this subgroup interact with phytochromes but – except for ERF55 and ERF58 – are expressed in seedlings or adult plants and not in seeds. Therefore, in the second work package, we aim to identify the functions of these AP2/ERFs in light signalling in seedlings and adult plants, using CRISPR/Cas9 generated erf higher order mutants and high-throughput screening for potential target genes by DAP-seq. Phytochromes bind to the highly conserved DNA-binding domain of AP2/ERFs, suggesting that binding to – and possibly regulation by – phytochromes may be widespread among AP2/ERFs. Several randomly picked AP2/ERFs from different subgroups indeed interacted with phytochromes. In the third work package, we therefore want to screen the entire AP2/ERF family for members interacting with phytochromes. In summary, we have identified members of the AP2/ERF transcription family as novel interactors of phytochromes. In the proposed project, we want to investigate their function in phytochrome-mediated light responses and elucidate the underlying molecular mechanisms.