Introduction

Our current understanding of the cellular and molecular mechanisms involved in the seasonal regulation of bud growth in woody trees in temperate regions is based mainly on vegetative bud studies (Goeckeritz & Hollender, 2021). In many trees, short days and decreasing temperature during late summer/early autumn induce the cessation of bud growth. The most visible sign of vegetative bud growth cessation is the formation of an apical bud, consisting of the shoot apical meristem and leaf primordia enclosed by protective bud scales (Nitsch, 1957; Goffinet & Larson, 1981; Ruttink et al., 2007; Kayal et al., 2011). When in a dormant state, meristems and leaf primordia inside vegetative buds become insensitive to growth promotion signals, and growth arrest is maintained by endogenous signals; this stage is described as endodormancy. Toward the end of winter, in a further dormant state, called ecodormancy, growth arrest is primarily maintained by low temperatures (Lang et al., 1987; Anderson, 2015). Finally, after dormancy release, ‘warm’ temperatures promote bud burst. This terminology and progression of events have been indifferently applied in Rosaceae species having vegetative and flower buds (e.g. Prunoidae species) or mixed buds (e.g. Pomoideae species). However, Hatch & Walker (1969) and Walker (1970) suggested that peach flower buds have different resting mechanisms from those of vegetative buds. In peach, chilling is essential for the proper differentiation of the floral reproductive whorls. An insufficient chilling exposure may lead to abortion of the reproductive whorls, low bud burst and nonuniform blooming, with a negative impact on fruit set and quality (Luna et al., 1991; Atkinson et al., 2013). During the chilling period, cytological observations indicated that the reproductive whorls differentiate very slowly in the bud, but the major developmental events, including ovule formation in the carpel and microsporogenesis and pollen maturation in the anthers, occur at the end of the chilling period. The onset of microsporogenesis is therefore considered a good cytological indicator of chilling requirement (CR) fulfillment (Reinoso et al., 2002; Julian et al., 2011; Ríos et al., 2013). However, despite these anatomical observations, physiological and molecular investigations have been performed considering the peach flower bud as a dormant bud (see references in Goeckeritz & Hollender, 2021).

Bud dormancy is regulated by hormonal, transcriptional, epigenetic and physiological changes (reviewed in Singh et al., 2017). Abscisic acid (ABA) concentrations are highest during deep endodormancy and decrease by ecodormancy in response to chilling, with a trend that is opposite to that of gibberellins (GAs), whereas the role of cytokinin (CK) as a regulator of dormancy appears to be less documented. Until now, at the genetic level, a group of six tandemly repeated transcription factors of the MADS-box gene family, named DORMANCY ASSOCIATED MADS-box genes (DAM1–6), were identified in the peach genome as potential markers of the dormancy (Bielenberg et al., 2008). First, the DAM genes were identified in the evergrowing (evg) peach mutant. The evg trait is genetically heritable and segregates as a single recessive gene (Rodriguez et al., 1994). The evg locus was then identified and mapped in a genomic region of 132 kb that was demonstrated to be partially deleted in four of the six clustered MADS-box genes. In evg, DAM1–DAM4 were physically deleted, and the expression levels of DAM5 and DAM6 were reduced (Bielenberg et al., 2004, 2008), whereas in wild-type peaches, DAM1, 4, 5 and 6 transcripts were downregulated in flower buds following dormancy release and differentially expressed in cultivars with different CRs (Leida et al., 2010, 2012). Low temperature was proposed to activate DAM transcription for dormancy induction by direct binding of the cold-dependent C-repeat binding factor (CBF) to DAM promoters (Saito et al., 2015; Niu et al., 2016; Zhao et al., 2018). The DAM genes were also identified in other species such as poplar (Ruttink et al., 2007), raspberry (Mazzitelli et al., 2007) and leafy spurge (Horvath et al., 2008). The six DAM genes of peach presumably originated from an ancestor related to the flowering transition regulator SHORT VEGETATIVE PHASE (SVP) of Arabidopsis thaliana, a transcriptional repressor that inhibits flowering by direct repression of the FLOWERING LOCUS T (FT) (Jiménez et al., 2009). In hybrid aspen, SVP has been characterized in vegetative buds as one of the major regulators of bud dormancy (Singh et al., 2018, 2019), but the functional role of SVP and DAM genes in Rosaceae remains to be elucidated.

The role of both epigenetic and chromatin regulation in DAM genes and bud dormancy has been studied in Rosaceae fruit trees. DNA methylation pattern variations were observed in sweet cherry flower buds in early winter (Rothkegel et al., 2020). In addition, it was observed that DAM genes are subject to chromatin regulation (Jimenéz et al., 2012; Leida et al., 2012; Niu et al., 2016). By considering possible commonalities between chromatin dynamics at the AtFLC locus and DAM gene loci during vernalization and winter bud dormancy, respectively, the distribution of chromatin marks, such as H3K4me3 and H3K27me3, were investigated in peach flower buds during endodormancy and ecodormancy by Zhu et al. (2020). They showed that expression of the five DAMs remains steadily unchanged with the ensuing warm temperature after chilling, and that this state of regulation correlates with robust increases of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in DAM4.

The regulation of both flower bud dormancy and bloom time in deciduous trees whose flower development spans the four seasons still requires more holistic studies. Understanding what regulates the physiological events occurring during flower bud dormancy and controls the fulfillment of CR is of particular importance in the context of global warming. Indeed, in recent years in milder regions, peach floral buds on trees do not always have their CR satisfied to complete development.

In this study, we focused our attention on flower bud development during winter in peach. To understand how bud development progression is regulated, we integrated cytological, epigenetic and chromatin genome-wide data with transcriptional outputs to obtain a complete picture of the main regulatory pathways involved in flower development during chilling accumulation. To reach this goal we change our paradigmatic view of the flower bud status from dormant to nondormant during the cold season. Our findings support the hypothesis that in peach flower buds the chilling accumulation allows flower differentiation to be completed.

中文翻译：

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桃花芽冷发育的染色质和转录动力学证据

介绍

我们目前对温带地区木本树木芽生长季节性调控所涉及的细胞和分子机制的理解主要基于营养芽研究 (Goeckeritz & Hollender,  2021 )。在许多树木中，夏末/初秋的短日照和温度下降会导致芽停止生长。营养芽生长停止最明显的迹象是顶芽的形成，由芽顶分生组织和被保护性芽鳞包围的叶原基组成（Nitsch，  1957 年；Goffinet 和 Larson，  1981 年；Ruttink等人，  2007 年；Kayal等,  2011). 当处于休眠状态时，营养芽内的分生组织和叶原基对生长促进信号变得不敏感，生长停滞由内源信号维持；这个阶段被描述为休眠期。在冬季即将结束时，处于更进一步的休眠状态，称为生态休眠，生长停滞主要由低温维持（Lang等人，  1987 年；Anderson，  2015 年）。最后，休眠解除后，“温暖”的温度会促进芽的萌发。这个术语和事件的进展已经无差别地应用于具有营养和花芽（例如Prunoidae种）或混合芽（例如Pomoideae种）的蔷薇科物种。然而，哈奇和沃克 (1969 ) 和 Walker ( 1970 ) 认为桃花芽与营养芽有不同的休眠机制。在桃子中，冷却对于花生殖轮的正确分化是必不可少的。低温暴露不足可能导致生殖轮流产、低芽爆发和不均匀开花，对坐果和质量产生负面影响（Luna等人，  1991 年；Atkinson等人，  2013 年）). 在冷却期，细胞学观察表明生殖轮在芽中分化非常缓慢，但主要的发育事件，包括心皮中的胚珠形成和花药中的小孢子发生和花粉成熟，发生在冷却期结束时。因此，小孢子发生的开始被认为是寒冷需求 (CR) 满足的良好细胞学指标（Reinoso等人，  2002 年；Julian等人，  2011 年；Ríos等人，  2013 年）). 然而，尽管有这些解剖学观察，生理学和分子学研究仍将桃花蕾视为休眠芽（参见 Goeckeritz & Hollender，  2021中的参考资料）。

芽休眠受激素、转录、表观遗传和生理变化的调节（综述于 Singh等人，  2017 年）。脱落酸 (ABA) 浓度在深度内休眠期间最高，生态休眠响应寒冷而降低，其趋势与赤霉素 (GAs) 相反，而细胞分裂素 (CK) 作为休眠调节剂的作用似乎是记录较少。到目前为止，在遗传水平上，一组六个串联重复的 MADS-box 基因家族转录因子，命名为 DORMANCY ASSOCIATED MADS-box 基因 (DAM1-6)，在桃基因组中被鉴定为休眠的潜在标记。 Bielenberg等人，  2008 年）。首先，DAM 基因在不断增长的( evg ) 桃突变体。evg性状在遗传上是可遗传的，并且分离为单个隐性基因（Rodriguez等人，  1994）。然后evg基因座被识别并映射到 132 kb 的基因组区域，该区域被证明在六个聚类 MADS-box 基因中的四个中被部分删除。在 e vg中，DAM1–DAM4 被物理删除，DAM5 和 DAM6 的表达水平降低（Bielenberg等人，  2004 年，2008 年）)，而在野生型桃中，DAM1、4、5 和 6 转录本在休眠释放后的花蕾中下调，并在具有不同 CR 的品种中差异表达 (Leida et al .,  2010 , 2012 )。低温被提议通过将冷依赖性 C 重复结合因子 (CBF) 直接结合到 DAM 启动子来激活 DAM 转录以诱导休眠（Saito等人，  2015 年；Niu等人，  2016 年；Zhao等人，  2018 年）。DAM 基因也在其他物种中得到鉴定，例如杨树（Ruttink等人，  2007 年）、覆盆子（Mazzitelli等人，  2007 年）和多叶大戟（Horvath等人，  2008 年）。桃的六个 DAM 基因可能起源于与拟南芥的开花过渡调节剂短营养期 (SVP) 相关的祖先，这是一种通过直接抑制 FLOWERING LOCUS T (FT) 来抑制开花的转录抑制因子（Jiménez等人，  2009 年）。在杂交白杨中，SVP 在营养芽中被描述为芽休眠的主要调节因子之一（Singh等人，  2018 年，2019 年），但 SVP 和 DAM 基因在蔷薇科中的功能作用仍有待阐明。

已经在蔷薇科果树中研究了表观遗传和染色质调控在 DAM 基因和芽休眠中的作用。在初冬的甜樱桃花蕾中观察到 DNA 甲基化模式变异 (Rothkegel et al .,  2020 )。此外，观察到 DAM 基因受染色质调控 (Jimenéz et al .,  2012 ; Leida et al . ,  2012 ; Niu et al .,  2016 )). Zhu等人分别考虑春化和冬芽休眠期间 AtFLC 基因座和 DAM 基因座染色质动力学之间可能存在的共性，研究了内休眠和生态休眠期间桃花蕾中 H3K4me3 和 H3K27me3 等染色质标记的分布. （2020 年）。他们表明，随着冷却后随之而来的温暖温度，五种 DAM 的表达保持稳定不变，并且这种调节状态与 sRNA 表达、H3K27me3 和 CHH 甲基化的强劲增加相关，这在 DAM4 中尤为明显。

花发育跨越四个季节的落叶乔木的花芽休眠和开花时间的调控仍然需要更全面的研究。了解是什么调节了花蕾休眠期间发生的生理事件并控制了 CR 的实现，这在全球变暖的背景下尤为重要。事实上，近年来在较温和的地区，树上的桃花芽并不总是满足其 CR 以完成发育。

在这项研究中，我们将注意力集中在桃子冬季花芽的发育上。为了解芽发育进程是如何被调控的，我们将细胞学、表观遗传学和染色质全基因组数据与转录输出相结合，以全面了解低温积累过程中花发育所涉及的主要调控途径。为实现这一目标，我们将寒冷季节花蕾状态的范式视图从休眠状态更改为非休眠状态。我们的研究结果支持这样的假设，即在桃花蕾中，低温积累允许完成花的分化。