Introduction

During the course of evolution, flowering plants have evolved different life history strategies, mostly related to their capacity to reproduce during their life cycle. From this perspective, most plants can be classified as monocarpic or polycarpic, depending on how many times they are able to undergo a reproductive phase during their lifetime. Annual plants are usually monocarpic, as they are able to complete their life cycle within a year, dying shortly after they have flowered and set seeds. Conversely, perennial plants are generally polycarpic, meaning that they will experience several reproductive phases during their lifetime, while they persist in the environment (Li et al., 2021). To achieve this, perennial plants usually show asynchronous development of meristems, which allows them to concentrate their reproductive effort only in certain parts, whereas the rest of the plant remains vegetative (Amasino, 2009). The model plant Arabis alpina presents a polycarpic perennial life habit. In addition, some accessions (e.g. Pajares) show a strong vernalization requirement, hence remaining vegetative until they experience prolonged periods of cold (Wang et al., 2009). The use of comparative studies between A. alpina and its annual relative Arabidopsis thaliana has allowed the identification of floral regulators with common but also distinct functions between these species, which can be indicative of differential adaption regarding life history strategies (Albani & Coupland, 2010).

Recent studies have identified key genes involved in the perennial flowering of A. alpina (Wang et al., 2009, 2011; Bergonzi et al., 2013; Hyun et al., 2019; Lázaro et al., 2019). One such core regulator of flowering in response to vernalization is PERPETUAL FLOWERING1 (PEP1), which is a MADS domain transcription factor and the orthologue of A. thaliana FLOWERING LOCUS C (FLC) (Wang et al., 2009). Similar to FLC in A. thaliana, vernalization results in the repression of PEP1 expression, which allows some meristems in the plant to transition into the reproductive phase and flower (Sheldon et al., 2000; Lázaro et al., 2018). After vernalization, PEP1 is upregulated, ensuring the maintenance of vegetative development in some axillary meristems and contributing to the overall perennial life history in A. alpina. Accordingly, pep1 mutant alleles show reduced life span in field experiments (Hughes et al., 2019).

Other examples of floral regulators with additional functions in A. alpina include factors that regulate the age-dependent response to vernalization, for example the A. alpina TERMINAL FLOWER 1 (AaTFL1), the APETALA2 transcription factors PEP2 and TARGET OF EAT2 (AaTOE2), and the A. alpina SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 15 (AaSPL15). These genes were shown to affect the age at which plants become competent to respond to flowering-inducing signals, the duration of vernalization they required for flowering and the maintenance of vegetative growth in axillary meristems (Wang et al., 2011; Hyun et al., 2019; Lázaro et al., 2019; Zhou et al., 2021). Despite its prominent role in vernalization, PEP1 is not the only regulator in this pathway as pep1 mutants still respond to prolonged cold treatments (Lázaro et al., 2018). In an effort to identify other regulators of flowering apart from PEP1, the pep1-1 mutant was used as background for an EMS (ethyl methanesulphonate) mutagenesis screen for enhancers of pep1 (eop; Zhou et al., 2021).

In this study we characterized three members of the eop mutant population, namely eop002, eop085 and eop091, which showed an extremely early flowering phenotype in long day (LD) conditions compared to pep1-1. These three mutants are allelic and all carry mutations in the coding sequence of a member of the AAA⁺ superfamily of ATPases, which we named FLOWERING REPRESSOR AAA⁺-ATPase 1 (AaFRAT1). By resequencing AaFRAT1 in our eop mutant collection, we detected 14 eop mutants carrying lesions in AaFRAT1, suggesting that it may play an instrumental role in the development of A. alpina. Furthermore, A. alpina lines carrying the Aafrat1 mutant alleles in the Pajares wild-type background, which requires vernalization to flower, showed compromised perennial traits and required shorter durations of vernalization to flower. At the shoot apical meristem, we detected AaFRAT1 mostly in the vasculature of young leaf primordia and very faintly at the rib zone. At the subcellular level, AaFRAT1 was localized at the interphase between endoplasmic reticulum (ER) tubules and peroxisomes (in seedling roots). In A. thaliana, the AaFRAT1 orthologue belongs to a cluster of four tandemly duplicated genes. Atfrat1 CRISPR mutants in A. thaliana showed very mild flowering time phenotypes, suggesting that the role of AtFRAT1 on flowering is not as prominent as that of AaFRAT1 in A. alpina. Our study is the first to demonstrate the involvement of a member of the AAA⁺ family of ATPases in flowering, which will open new pathways for future research.

中文翻译：

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FLOWERING REPRESSOR AAA+ ATPase 1 是一种新的高山阿拉伯多年生开花调控因子

介绍

在进化过程中，开花植物进化出不同的生命史策略，主要与它们在生命周期中的繁殖能力有关。从这个角度来看，大多数植物可以分为单果或多果，这取决于它们在其一生中能够经历多少次生殖阶段。一年生植物通常是单结实的，因为它们能够在一年内完成其生命周期，在开花和结籽后不久就死亡。相反，多年生植物通常是多果的，这意味着它们在其一生中会经历几个生殖阶段，同时它们会在环境中持续存在（Li et al ., 2021）。为了实现这一点，多年生植物通常表现出分生组织的不同步发育，这使得它们只能将其生殖努力集中在某些部分，而植物的其余部分保持营养状态（Amasino，  2009 年）。模式植物Arabis alpina具有多年生多果的生活习性。此外，一些种质（例如 Pajares）表现出强烈的春化要求，因此在经历长时间的寒冷之前保持植物生长（Wang等人，2009 年）。使用A. alpina与其年度相关拟南芥之间的比较研究已经允许鉴定在这些物种之间具有共同但也具有不同功能的花卉调节剂，这可以表明对生活史策略的不同适应（Albani＆Coupland，  2010）。

最近的研究已经确定了参与A. alpina多年生开花的关键基因（Wang等人，2009 年，2011 年；Bergonzi等人，2013 年；Hyun等人，2019 年；Lázaro等人，2019 年）。一种响应春化的开花的核心调节因子是PERPETUAL FLOWERING1 ( PEP1 )，它是一种 MADS 结构域转录因子，是拟南芥 FLOWERING LOCUS C ( FLC ) 的直系同源物 (Wang et al ., 2009 )。类似于FLC在拟南芥中，春化导致PEP1表达的抑制，这允许植物中的一些分生组织过渡到生殖阶段和开花（Sheldon等人，2000 年；Lázaro等人，2018 年）。春化后，PEP1被上调，确保一些腋生分生组织的营养发育得以维持，并有助于A. alpina的整体多年生生活史。因此，pep1突变等位基因在田间实验中显示寿命缩短（Hughes等人，2019 年）。

在A. alpina中具有附加功能的花调节剂的其他例子包括调节对春化的年龄依赖性反应的因子，例如A. alpina TERMINAL FLOWER 1 ( AaTFL1 )、APETALA2转录因子PEP2和TARGET OF EAT2 ( AaTOE2 )，和A. alpina SQUAMOSA 启动子结合蛋白样 15 ( AaSPL15 )。这些基因显示出影响植物能够对开花诱导信号作出反应的年龄、开花所需的春化持续时间和腋生分生组织营养生长的维持（Wang等人，., 2011 ; Hyun等人，2019 年；Lázaro等人，2019 年；周等人，2021）。尽管 PEP1 在春化过程中发挥着重要作用，但 PEP1并不是该途径中的唯一调节因子，因为pep1突变体仍然对长时间的冷处理有反应（Lázaro等人，2018 年）。为了鉴定除PEP1之外的其他开花调节因子，pep1-1突变体被用作pep1 增强子( eop ; Zhou等人，2021 年）。

在这项研究中，我们表征了eop突变体群体的三个成员，即eop002、eop085和eop091 ，与pep1-1相比，它们在长日照( LD) 条件下表现出极早的开花表型。^{这三个突变体是等位基因的，并且都在 ATP 酶 AAA +}超家族成员的编码序列中携带突变，我们将其命名为FLOWERING REPRESSOR AAA ⁺ -ATPase 1 ( AaFRAT1 )。通过对我们的eop突变体集合中的AaFRAT1重新测序，我们检测到 14 个eop突变体在AaFRAT1，表明它可能在A. alpina的发育中发挥重要作用。此外，在 Pajares 野生型背景中携带Aafrat1突变等位基因的A. alpina品系，需要春化才能开花，显示出受损的多年生性状并且需要更短的春化时间才能开花。在茎尖分生组织，我们主要在幼叶原基的脉管系统中检测到 AaFRAT1，在肋区非常微弱。在亚细胞水平上，AaFRAT1 定位于内质网 (ER) 小管和过氧化物酶体之间的相间（在幼苗根中）。在拟南芥中，AaFRAT1 直系同源物属于四个串联重复基因的簇。Atfrat1拟南芥中的 CRISPR 突变体表现出非常温和的开花时间表型，这表明 AtFRAT1 对开花的作用不如A. alpina中的 AaFRAT1 突出。我们的研究首次证明了 AAA ⁺ ATP 酶家族成员参与开花，这将为未来的研究开辟新的途径。