FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple
FLOWERING LOCUS T1 和 TERMINAL FLOWER1 调控网络介导苹果开花起始

Abstract 抽象的

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX–LOOP–HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.
花芽形成是直接决定果树产量和果实品质的关键过程。花诱导是通过两种开花相关蛋白 FLOWERING LOCUS T (FT) 和 TERMINAL FLOWER1 (TFL1) 之间的平衡来调节的；然而，建立和维持这种动态平衡的机制在很大程度上仍然难以捉摸。在这里，我们发现，在苹果 ( Malus × Domestica Borkh.) 中， MdFT1主要在短芽中表达，并且在花诱导期间表达量增加；相反， MdTFL1在花诱导过程中在顶端表现出下调，表明 MdTFL1 在花抑制中发挥作用。有趣的是， MdFT1和MdTFL1转录本均受转录因子碱性HELIX–LOOP–HELIX48 (MdbHLH48)直接调控，并且MdbHLH48在拟南芥( Arabidopsis thaliana )和番茄( Solanum lycopersicum )中的过度表达会导致加速开花。结合和激活分析表明，MdbHLH48 充当MdFT1的正调节因子和MdTFL1的负调节因子。进一步的研究表明，MdFT1和MdTFL1都与MdWRKY6蛋白竞争性相互作用，分别促进和抑制MdWRKY6介导的靶基因APPLE FLORIICAULA / LFY （ AFL1 ，苹果LEAFY样基因）的转录激活，最终调节苹果花芽的形成。这些发现说明了 MdbHLH48-MdFT1/MdTFL1-MdWRKY6 模块对开花的微调调节，并为苹果花芽形成提供了见解。

Introduction 介绍

Flowering is a key event in the plant life cycle that determines yield and fruit characteristics. Perennial woody fruit trees differ greatly in their flowering habits relative to annual herbaceous plants. Apple (Malus × domestica Borkh.) trees must pass through a relatively long juvenile phase (4 to 8 yr or more) before attaining the ability to flower, which severely limits the efficiency of fruit tree breeding and profitability. The adult apple trees generate flower buds annually, and the entire flowering process typically spans 2 growing seasons (Foster et al. 2003; Hanke et al. 2007; Dadpour et al. 2011; Mimida et al. 2013). Specifically, flower induction, initiation, and flower differentiation take place in summer and autumn of the preceding year and bloom in the following spring. Notably, flower induction is a crucial process, as its obstruction will lead to a reduction in the number of flower buds, subsequently impacting the apple quality and yield in the ensuing year (Buban and Faust 1982). The occurrence of alternate bearing, a prominent concern in breeding and cultivation, is directly linked to the impairment of flower bud formation, resulting in yield fluctuations. Therefore, to ensure stability in fruit yield and quality, it is imperative to achieve consistent and abundant floral bud formation during the preceding summer. An improved understanding of the molecular basis of floral induction is of great importance for improving breeding efficiency and maintaining a steady harvest in apple.
开花是植物生命周期中的一个关键事件，决定产量和果实特性。多年生木本果树的开花习性与一年生草本植物有很大不同。苹果( Malus × Domestica Borkh.)树必须经过较长的幼年期(4～8年甚至更长)才能开花，这严重限制了果树育种的效率和盈利能力。成年苹果树每年都会产生花芽，整个开花过程通常跨越2个生长季节（ Foster等人，2003年； Hanke等人，2007年； Dadpour等人，2011年； Mimida等人，2013年）。具体而言，花的诱导、起始和花分化发生在前一年的夏季和秋季，并在来年的春季开花。值得注意的是，花诱导是一个至关重要的过程，因为它的阻碍将导致花芽数量减少，从而影响来年的苹果质量和产量（ Buban和Faust 1982 ）。交替结果的发生是育种和栽培中的一个突出问题，它与花芽形成受损直接相关，导致产量波动。因此，为保证果实产量和品质的稳定，必须在前年夏季实现一致、丰富的花芽形成。加深对花诱导分子基础的了解对于提高育种效率和保持苹果的稳定收获具有重要意义。

Flower induction is the process of transforming vegetative buds into reproductive buds. This process is determined by a complex interplay of 4 major interconnected pathways, namely, the photoperiod, vernalization, gibberellic acid, and autonomous pathways. The final outputs of all pathways converge on a few key flowering integrator genes, one notable one being FLOWERING LOCUS T (FT; Putterill et al. 2004; Amasino and Michaels 2010). The FT protein is thought as at least a part of the florigen signal that moves to the shoot apical meristem (SAM) to trigger floral initiation (Corbesier et al. 2007; Jaeger and Wigge 2007). FT belongs to the phosphatidylethanolamine-binding protein (PEBP) family, which plays a pivotal role in regulating the seasonal onset of reproductive development and the transition from branch to floral fate in inflorescence primordia (Higuchi 2018). Another well-characterized PEBP protein with an opposite function to FT, named TERMINAL FLOWER1 (TFL1), has been identified as an antiflorigen; the protein can move short distances to block the floral transition in the main apex (Bradley et al. 1997; Conti and Bradley 2007; Jaeger et al. 2013). Both TFL1 and FT have been shown to physically interact with the basic leucine zipper domain (bZIP) transcription factor FD, a key regulator of flowering time at the SAM. The TFL1-FD and FT-FD complex subsequently regulate multiple floral meristem-specific genes, including LEAFY (LFY), APETALA1 (AP1), and FRUITFULL (FUL), thereby affecting the transition to reproductive growth (Abe et al. 2005; Wigge et al. 2005; Hanano and Goto 2011; Randoux et al. 2014; Kaneko-Suzuki et al. 2018; Collani et al. 2019; Zhu et al. 2020).
花诱导是将营养芽转变为生殖芽的过程。这一过程是由 4 个主要相互关联途径（即光周期、春化、赤霉酸和自主途径）复杂的相互作用决定的。所有途径的最终输出都集中在一些关键的开花整合基因上，其中一个值得注意的基因是FLOWERING LOCUS T （ FT ； Putterill 等人，2004 年； Amasino 和 Michaels，2010 年）。 FT 蛋白被认为至少是成花素信号的一部分，该信号移动到茎尖分生组织（SAM）以触发花的起始（ Corbesier 等人，2007 年； Jaeger 和 Wigge，2007 年）。 FT 属于磷脂酰乙醇胺结合蛋白（PEBP）家族，在调节花序原基生殖发育的季节开始和从分枝到花命运的转变中发挥着关键作用（ Higuchi 2018 ）。另一种特征明确的 PEBP 蛋白，其功能与 FT 相反，名为 TERMINAL FLOWER1 (TFL1)，已被鉴定为抗花素；该蛋白质可以短距离移动以阻止主尖的花转变（ Bradley et al. 1997 ； Conti and Bradley 2007 ； Jaeger et al. 2013 ）。 TFL1 和 FT 均已被证明与碱性亮氨酸拉链结构域 (bZIP) 转录因子 FD 发生物理相互作用，FD 是 SAM 开花时间的关键调节因子。 TFL1-FD 和 FT-FD 复合物随后调节多个花分生组织特异性基因，包括LEAFY ( LFY )、 APETALA1 ( AP1 ) 和FRUITFULL ( FUL )，从而影响向生殖生长的过渡。 2005年；维格等人。 2005年；花野和后藤 2011 ；朗杜等人。 2014年；金子铃木等人。 2018年；科拉尼等人。 2019年；朱等人。 2020 ）。

FT-like genes and TFL1-like genes have been isolated and functionally analyzed in numerous woody perennial plants, such as pear (Pyrus communis L.), apple, citrus (Citrus sinensis L. Osbeck), and kiwifruit (Actinidia spp.; Pillitteri et al. 2004; Endo et al. 2005; Kotoda et al. 2010; Freiman et al. 2012; Varkonyi-Gasic et al. 2013; Wu et al. 2022). The apple genome contains 2 homologs of FT (MdFT1/MdFT2) and 2 homologs of TFL1 (MdTFL1/MdTFL1a; Kotoda and Wada 2005; Mimida et al. 2009; Kotoda et al. 2010). Given that the expression of MdFT1, but not MdFT2, was predominantly in apical buds and exhibits a gradual increase within the apex during the expected floral induction period (Hättasch et al. 2008; Kotoda et al. 2010; Guitton et al. 2016), it is likely that MdFT1 accelerates flowering. Indeed, overexpression of MdFT1 induces precocious flowering in apple (Kotoda et al. 2010; Tränkner et al. 2010). Conversely, MdTFL1/MdTFL1a, which encodes a floral repressor, displays a rapid decline in expression within the apices either prior to or during the floral induction and initiation periods (Hättasch et al. 2008; Mimida et al. 2009, 2011b). Transgenic knockdown of TFL1 genes in apple (MdTFL1) and pear (PcTFL1) results in precocious flowering (no juvenile phase) and terminal single flowers (Kotoda et al. 2006; Flachowsky et al. 2012; Freiman et al. 2012). Collectively, these findings suggest that MdFT1 antagonizes MdTFL1 in regulating the phase transition and may exert an influence on floral development throughout the growing season in apple. It is thus clear that the expression levels of MdFT1 and MdTFL1 appear to be pivotal to the function of their products, and the balance of MdTFL1 and MdFT1 ratios plays a decisive role in determining whether the developing buds will ultimately give rise to flower buds or remain vegetative. However, the mechanisms underlying the establishment and maintenance of the dynamic and intricate expression of MdFT1 and MdTFL1 remain elusive.

Multiple transcription factors, including the basic HELIX–LOOP–HELIX (bHLH), WRKY, MYB, and the NUCLEAR FACTOR Y (NF-Y), have been implicated in the regulation of flowering (Li et al. 2017; Zhou et al. 2022; Wang et al. 2023a). The bHLH transcription factors are one of the most extensively characterized transcription factor families in plants, and increasing evidence indicates that bHLH transcription factors are involved in flowering regulation. For example, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a class of bHLH proteins, accelerates flowering by directly activating FT expression under high-temperature conditions (Kumar et al. 2016). CRYPTOCHROME-INTERACTING bHLH (CIBs), which belong to BEE/CIB subfamily of bHLH transcription factor, specifically interact with cryptochrome2 (CRY2) in response to blue light and regulate floral initiation by activating the transcription of FT (Liu et al. 2013). Arabidopsis (Arabidopsis thaliana) bHLH48 and bHLH60 positively modulate GA-mediated flowering through activation of FT expression (Li et al. 2017). Normally, bHLH transcription factors interact to form homo- or heterodimers that regulate the expression of target genes by binding to E-box (CANNTG) or G-box (CACGTG) motifs within target genes (Hao et al. 2021). In addition, several lines of evidence have suggested WRKY transcription factors also play a role in floral initiation (Wang et al. 2023a). For example, WRKY34 is involved in vernalization-mediated flowering by modulating CULLIN3A (CUL3A) expression (Hu et al. 2014), and WRKY71 and WRKY75 accelerate flowering by binding directly to the W-box motif in the promoters of FT and LFY to activate their expression (Yu et al. 2016; Zhang et al. 2018). WRKY12 and WRKY13 exert opposing effects on flowering time under noninduced short-day conditions via the direct regulation of FUL (Li et al. 2016). However, in apple, relatively few studies have explored the role and mechanism of these transcription factors in the regulation of apple flowering.
多种转录因子，包括基本的 HELIX-LOOP-HELIX (bHLH)、WRKY、MYB 和核因子 Y (NF-Y)，与开花调节有关（ Li et al. 2017 ； Zhou et al. 2017）。 2022 ；王等人，2023a ）。 bHLH 转录因子是植物中最广泛表征的转录因子家族之一，越来越多的证据表明 bHLH 转录因子参与开花调节。例如，一类 bHLH 蛋白 PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) 在高温条件下通过直接激活FT表达来加速开花 ( Kumar et al. 2016 )。隐花色素相互作用bHLH（CIB）属于bHLH转录因子的BEE/CIB亚家族，在蓝光响应下与隐花色素2（CRY2）特异性相互作用，并通过激活FT的转录来调节花的起始（ Liu et al. 2013 ）。拟南芥 ( Arabidopsis thaliana ) bHLH48 和 bHLH60 通过激活FT表达正向调节 GA 介导的开花 ( Li et al. 2017 )。通常，bHLH 转录因子相互作用形成同二聚体或异二聚体，通过与靶基因内的 E-box (CANNTG) 或 G-box (CACGTG) 基序结合来调节靶基因的表达 ( Hao et al. 2021 )。此外，一些证据表明 WRKY 转录因子也在花起始中发挥作用（ Wang et al. 2023a ）。例如，WRKY34 通过调节CULLIN3A ( CUL3A ) 表达参与春化介导的开花（ Hu et al. 2014），WRKY71和WRKY75通过直接结合FT和LFY启动子中的W-box基序来激活其表达来加速开花（ Yu等人，2016 ； Zhang等人，2018 ）。在非诱导短日照条件下，WRKY12 和 WRKY13 通过直接调节FUL对开花时间产生相反的影响（ Li et al. 2016 ）。然而，在苹果中，探索这些转录因子在苹果开花调控中的作用和机制的研究相对较少。

In this study, apple MdFT1 and MdTFL1 showed opposite expression patterns during the floral induction stage. Interestingly, we demonstrated that the expression of MdFT1 and MdTFL1 is differentially regulated by MdbHLH48, which positively regulates flowering in transgenic Arabidopsis and tomato (Solanum lycopersicum). Further analysis indicated that MdTFL1 may possibly compete with MdFT1 for interaction with MdWRKY6 transcription factor and affects the MdWRKY6 transcriptional activity on its downstream target gene AFL1, which in turn affects flowering. Thus, our work provides insights into flowering initiation mediated by the regulatory networks of FT and TFL1 in apple.
在本研究中，苹果MdFT1和MdTFL1在花诱导阶段表现出相反的表达模式。有趣的是，我们证明MdFT1和MdTFL1的表达受到MdbHLH48的差异调节，MdbHLH48对转基因拟南芥和番茄（ Solanum lycopersicum ）的开花有正向调控。进一步分析表明，MdTFL1可能与MdFT1竞争与MdWRKY6转录因子的相互作用，影响MdWRKY6对其下游靶基因AFL1的转录活性，进而影响开花。因此，我们的工作提供了对苹果中 FT 和 TFL1 调控网络介导的开花起始的见解。

Results 结果

The expression patterns of MdFT1 and MdTFL1 in apical buds during flower transition in apple
苹果花期顶芽中MdFT1和MdTFL1的表达模式

To gain insight into the role of apple FT-like proteins (MdFT1/MdFT2) and TFL1-like proteins (MdTFL1/MdTFL1a), we analyzed the expression patterns of their genes in various organs of plants during flower transition stages (Fig. 1; Supplementary Fig. S1). MdFT1 转录本主要积累在顶芽中，并在花诱导过程中逐渐增加。相反，它在根、茎、花和果组织中的存在很少. Notably, the transcript abundance of MdFT1 in the mature leaves was comparatively lower than that detected in apical buds. In contrast, MdFT2 was primarily detected in stems and young fruits, with partial presence in mature leaves and a weak expression in apical buds during floral induction (Supplementary Fig. S1), suggesting that MdFT2 may play a role in processes other than flowering regulation. In addition, we analyzed the expression levels of MdFT1 and MdFT2 in the apical buds from ‘Fuji’ trees during floral induction using RNA sequencing (RNA-seq). The analysis of RNA-seq read counts (Supplementary Fig. S2A) and reverse transcription quantitative PCR (RT-qPCR) analysis (Supplementary Fig. S2B) indicated that MdFT1 exhibited moderate expression while MdFT2 showed very low expression levels in the shoot apex, which is consistent with previous findings (Kotoda et al. 2010; Zhang et al. 2019).
为了深入了解苹果FT类蛋白（MdFT1/MdFT2）和TFL1类蛋白（MdTFL1/MdTFL1a）的作用，我们分析了它们的基因在花转变阶段植物各器官中的表达模式（图1 ；补充图S1 ）。 MdFT1分解本主要积累在顶芽中，并在花感应过程中逐渐增加。相反，它在根、茎、花和果组织中的存在很少。值得注意的是，成熟叶片中MdFT1的转录丰度相对低于顶芽中检测到的转录丰度。相比之下， MdFT2主要在茎和幼果中检测到，在成熟叶片中部分存在，并且在花诱导期间在顶芽中表达较弱（补充图S1 ），这表明 MdFT2 可能在开花调节以外的过程中发挥作用。此外，我们使用 RNA 测序 (RNA-seq) 分析了花诱导过程中“富士”树顶芽中MdFT1和MdFT2的表达水平。 RNA-seq读数计数分析（补充图S2A ）和逆转录定量PCR（RT-qPCR）分析（补充图S2B ）表明MdFT1在茎尖表现出中等表达，而MdFT2在茎尖表现出非常低的表达水平，这与之前的发现一致（ Kotoda et al. 2010 ； Zhang et al. 2019 ）。

On the other hand, both MdTFL1 and MdTFL1a were additionally detected in roots, phloem of stems, and apical buds during the early stage of flower induction. Our investigation of RNA-seq read counts (Supplementary Fig. S2A), and RT-qPCR analysis (Supplementary Fig. S2B), suggested that the expression level of MdTFL1a was relatively lower than that of MdTFL1 in the shoot apex.
另一方面，在花诱导的早期阶段，在根、茎的韧皮部和顶芽中另外检测到MdTFL1和MdTFL1a 。我们对 RNA-seq 读数计数（补充图 S2A ）和 RT-qPCR 分析（补充图 S2B ）的研究表明，茎尖中MdTFL1a的表达水平相对低于MdTFL1 。

Apple flower buds are often initiated on the terminal buds of short shoots (also called spurs), while flower buds are formed much less frequently on lateral buds of vegetative shoots (1-yr elongated shoots; Fig. 1, A and B; Hanke et al. 2007). To determine the morphological changes of apical meristems of the 2 types of buds, sections of buds were prepared (Fig. 1C). The meristem in spur buds changed into a floral meristem (at 120 d after full bloom [DAFB]). In contrast, the meristem in lateral buds was narrow and remained vegetative at 120 DAFB. MdFT1 was relatively more highly expressed in spur buds than in lateral buds of 1-yr elongated shoots, while the expression level of MdFT2 was only high at 30 DAFB in spur buds (Fig. 1D). In contrast, MdTFL1 and MdTFL1a were more highly expressed in lateral buds than in spur buds, and MdTFL1 expression decreased rapidly during flower induction, with an opposing expression pattern to that of MdFT1 (Fig. 1D). Moreover, higher expression levels of flower genes MdAP1 and MdAFL1 in spur buds were observed (Fig. 1D), supporting the view that spur buds have a greater probability of developing into flower buds.
苹果花芽通常在短芽（也称为短枝）的顶芽上萌生，而花芽在营养芽的侧芽上形成的频率要低得多（1年伸长芽；图1，A和B ； Hanke等等2007 ）。为了确定两种芽的顶端分生组织的形态变化，制备了芽的切片（图1C ）。短芽中的分生组织转变为花分生组织（盛花后120天[DAFB]）。相反，侧芽的分生组织较窄，在 120 DAFB 时仍保持营养状态。在1年伸长芽中， MdFT1在直芽中的表达量相对高于在侧芽中的表达量，而MdFT2的表达水平仅在30DAFB时在直芽中较高(图1D )。相反， MdTFL1和MdTFL1a在侧芽中的表达高于在短芽中的表达，并且MdTFL1的表达在花诱导过程中迅速下降，与MdFT1的表达模式相反(图1D )。此外，在直芽中观察到花基因MdAP1和MdAFL1的表达水平较高(图1D )，支持了直芽发育成花芽的可能性更大的观点。

Collectively, these observations strongly support previous studies (Hättasch et al. 2008; Mimida et al. 2009; Kotoda et al. 2010; Mimida et al. 2011b; Guitton et al. 2016; Zhang et al. 2019) implicating that the expression levels of MdFT1 and MdTFL1 are closely related to floral induction, with high MdFT1 expression favoring floral induction and high MdTFL1 expression not favoring or inhibiting floral induction.
总的来说，这些观察结果强烈支持了之前的研究（ Hättasch et al. 2008 ； Mimida et al. 2009 ； Kotoda et al. 2010 ； Mimida et al. 2011b ； Guitton et al. 2016 ； Zhang et al. 2019 ），表明表达水平MdFT1和MdTFL1的表达与花诱导密切相关， MdFT1高表达有利于花诱导，而MdTFL1高表达不利于或抑制花诱导。

MdbHLH48 directly binds to both MdFT1 and MdTFL1 promoters and regulates their expression
MdbHLH48 直接结合MdFT1和MdTFL1启动子并调节其表达

To understand the regulatory mechanism governing expression of MdFT1 and MdTFL1, the Jasper (https://jaspar.elixir.no/) and PlantTFDB (http://planttfdb.gao-lab.org/blast.php) online tools were used to predict possible upstream transcription factors, and several bHLH transcription factors were identified, including bHLH48, which is involved in regulating floral transition by activating FT transcription in Arabidopsis (Li et al. 2017). Moreover, we previously used the MdTFL1 promoter fragment (−898 to −503 bp) as bait to screen a yeast 1-hybrid (Y1H) cDNA library derived from ‘Fuji’ apple buds, and 2 bHLH transcription factors, bHLH48 (MD14G1064200) and bHLH105 (MD11G1227800), were identified (Supplementary Table S1). Considering that Arabidopsis bHLH48 can bind to FT promoter (Li et al. 2017), we speculate that MdbHLH48 (a homolog of Arabidopsis bHLH48) may be a common upstream regulator of MdTFL1 and MdFT1 in apple. In order to verify this conjecture, we first scanned the MdFT1 and MdTFL1 promoters (2,000 bp upstream from the start codon) and found 7 and 8 E-box (CANNTG) motifs (bHLH-binding motif; Hao et al. 2021) in MdFT1 and MdTFL1 promoters, respectively (Supplementary Fig. S3). Next, we examined whether apple MdbHLH48 could directly bind to the promoters of MdFT1 and MdTFL1 using Y1H assays. The results showed that MdbHLH48 could bind to promoter fragments of MdFT1 (pMdFT1-F1 and pMdFT1-F2) and promoter fragments of MdTFL1 (pMdTFL1-F1 and pMdTFL1-F2) containing bHLH-binding E-box motifs (Fig. 2A). In addition, electrophoretic mobility shift assays (EMSAs) further verified that MdbHLH48 specifically binds to MdFT1 and MdTFL1 probes containing E-box motif, and that when E-box was mutated, the binding was not observed (Fig. 2B). These results indicate that MdbHLH48 can directly associate with both MdFT1 and MdTFL1 promoters via the E-box motif.
为了了解MdFT1和MdTFL1表达的调控机制，使用 Jasper ( https://jaspar.elixir.no/ ) 和 PlantTFDB ( http://planttfdb.gao-lab.org/blast.php ) 在线工具预测可能的上游转录因子，并鉴定了几个 bHLH 转录因子，包括 bHLH48，它在拟南芥中通过激活FT转录来参与调节花转变（ Li et al. 2017 ）。此外，我们之前使用MdTFL1启动子片段（-898至-503 bp）作为诱饵筛选源自'Fuji'苹果芽的酵母1-杂交（Y1H）cDNA文库，以及2个bHLH转录因子bHLH48（MD14G1064200）和鉴定出bHLH105（MD11G1227800）（补充表S1 ）。考虑到拟南芥bHLH48可以结合FT启动子( Li et al. 2017 )，我们推测MdbHLH48(拟南芥bHLH48的同源物)可能是苹果中MdTFL1和MdFT1的常见上游调节因子。为了验证这个猜想，我们首先扫描了MdFT1和MdTFL1启动子（距起始密码子上游2,000 bp），并在MdFT1中发现了7个和8个E-box（CANNTG）基序（bHLH结合基序； Hao et al. 2021 ）和MdTFL1启动子，分别（补充图S3 ）。接下来，我们使用 Y1H 测定检查了苹果 MdbHLH48 是否可以直接结合MdFT1和MdTFL1的启动子。 结果显示，MdbHLH48可以结合MdFT1的启动子片段( pMdFT1-F1和pMdFT1-F2 )和含有bHLH结合E-box基序的MdTFL1的启动子片段( pMdTFL1-F1和pMdTFL1-F2 )(图2A )。此外，电泳迁移率变动分析(EMSA)进一步证实MdbHLH48特异性结合含有E-box基序的MdFT1和MdTFL1探针，并且当E-box突变时，没有观察到结合(图2B )。这些结果表明 MdbHLH48 可以通过 E-box 基序直接与MdFT1和MdTFL1启动子结合。

To investigate how MdbHLH48 regulates MdFT1 and MdTFL1, a dual-luciferase (LUC) reporter assay was performed in Nicotiana benthamiana leaves (Fig. 2C). The MdFT1 and MdTFL1 promoters were fused with LUC as the reporter (pMdFT1-LUC and pMdTFL1-LUC), and the highly expressed MdbHLH48 (35S::MdbHLH48) was used as the effector. When 35S::MdbHLH48 was cotransformed with pMdFT1-LUC, MdFT1 promoter activity increased significantly, while coexpression of MdbHLH48 with pMdTFL1-LUC repressed MdTFL1 promoter activity (Fig. 2C). Thus, MdbHLH48 appears to directly activate the expression of MdFT1 but repress MdTFL1.
为了研究MdbHLH48如何调节MdFT1和MdTFL1 ，在本塞姆氏烟草叶子中进行了双荧光素酶（LUC）报告基因测定（图2C ）。 MdFT1和MdTFL1启动子与LUC融合作为报告基因（ pMdFT1-LUC和pMdTFL1-LUC ），并使用高表达的MdbHLH48 （ 35S::MdbHLH48 ）作为效应子。当35S::MdbHLH48与pMdFT1-LUC共转化时， MdFT1启动子活性显着增加，而MdbHLH48与pMdTFL1-LUC共表达抑制MdTFL1启动子活性(图2C )。因此，MdbHLH48似乎直接激活MdFT1的表达但抑制MdTFL1 。

To further investigate the relationship between MdbHLH48 and MdFT1/MdTFL1 in apple, we overexpressed MdbHLH48 (35S::MdbHLH48-GFP) in apple calli, with empty GFP serving as a negative control (Fig. 2D; Supplementary Fig. S4, A and B). The overexpression of MdbHLH48 considerably upregulated MdFT1 expression in apple calli relative to the MdFT1 expression level in the control calli (empty vector) under the same conditions (Fig. 2D), demonstrating that MdbHLH48 induces MdFT1 expression. However, the expression of MdTFL1 could not be detected either in transgenic or control calli (Supplementary Fig. S4C). In addition, a GUS transactivation assay was performed in apple leaves to further verify transcriptional regulation through coexpressing 35S::MdbHLH48 and MdFT1/MdTFL1 promoter-fused GUS reporters (pMdFT1::GUS and pMdTFL1::GUS). The apple leaves containing pMdFT1::GUS and 35S::MdbHLH48 exhibited significantly higher GUS enzyme activity than those harboring pMdFT1::GUS alone (Fig. 2E). On the contrary, the apple leaves containing pMdTFL1::GUS and 35S::MdbHLH48 exhibited lower GUS enzyme activity than those harboring pMdTFL1::GUS alone (Fig. 2E). Taken together, these data demonstrate that MdbHLH48, as a common regulator of MdFT1 and MdTFL1, activates the transcription of the MdFT1 gene and represses the transcription of MdTFL1 in apple.
为了进一步研究苹果中MdbHLH48和MdFT1 / MdTFL1之间的关系，我们在苹果愈伤组织中过表达MdbHLH48 （ 35S::MdbHLH48-GFP ），并以空GFP作为阴性对照（图2D ；补充图S4，A和B） ）。相对于相同条件下对照愈伤组织(空载体)中的MdFT1表达水平， MdbHLH48的过表达显着上调苹果愈伤组织中的MdFT1表达(图2D )，证明MdbHLH48诱导MdFT1表达。然而，在转基因或对照愈伤组织中均未检测到MdTFL1的表达（补充图 S4C ）。此外，在苹果叶中进行了 GUS 反式激活测定，通过共表达35S::MdbHLH48和MdFT1 / MdTFL1启动子融合的 GUS 报告基因（ pMdFT1::GUS和pMdTFL1::GUS ）进一步验证转录调控。含有pMdFT1::GUS和35S::MdbHLH48的苹果叶表现出比单独含有pMdFT1::GUS的苹果叶显着更高的GUS酶活性（图2E ）。相反，含有pMdTFL1::GUS和35S::MdbHLH48的苹果叶表现出比单独含有pMdTFL1::GUS的苹果叶更低的GUS酶活性（图2E ）。 总之，这些数据证明MdbHLH48作为MdFT1和MdTFL1的共同调节因子，在苹果中激活MdFT1基因的转录并抑制MdTFL1的转录。

Overexpression of MdbHLH48 caused early flowering
MdbHLH48过表达导致早花

MdbHLH48 contains a bHLH domain and has a close relationship with Arabidopsis AtbHLH48 (belongs to bHLH subgroup XII; Fig. 3, A and B). MdbHLH48 was highly expressed in the leaves and particularly prominent in apical buds, but low expression was detected in fruit (Fig. 3C). MdbHLH48 expression was higher in spur buds than in lateral buds, and gradually increased during floral transition (Fig. 3D), showing the same expression pattern as MdFT1 but opposite to that of MdTFL1 (Fig. 1D). Transient expression in N. benthamiana leaves of 35S::MdbHLH48-GFP showed green fluorescence in the nucleus, overlapping with red fluorescence of the mCherry nuclear marker, indicating that MdbHLH48 is a nuclear-localized protein (Fig. 3E).
MdbHLH48含有bHLH结构域，与拟南芥AtbHLH48有密切关系（属于bHLH亚组XII；图3，A和B ）。 MdbHLH48在叶子中高表达，在顶芽中尤其突出，但在果实中检测到低表达(图3C )。 MdbHLH48在直芽中的表达量高于侧芽，并在花转变期间逐渐增加(图3D )，表现出与MdFT1相同但与MdTFL1相反的表达模式(图1D )。 35S::MdbHLH48 - GFP在本塞姆氏烟草叶子中的瞬时表达显示出细胞核中的绿色荧光，与mCherry核标记的红色荧光重叠，表明MdbHLH48是核定位蛋白(图3E )。

To establish whether MdbHLH48 affects flowering transition, we generated transgenic Arabidopsis plants overexpressing 35S::bHLH48-GFP in Arabidopsis Col-0. Consequently, 3 35S::MdbHLH48 lines (#1, #3, and #8) with significant expression were selected for further analysis (Fig. 3G; Supplementary Fig. S4D). The 35S::MdbHLH48 lines displayed an obvious early-flowering phenotype under long-day (16-h light/8-h dark) conditions (Fig. 3, F and H). Moreover, we found that the expression of AtFT was significantly elevated; in contrast, AtTFL1 was slightly suppressed in the 35S::MdbHLH48 Arabidopsis seedlings (Supplementary Fig. S4C). The expression levels of several flowering-promoted genes including AtLFY, AtAP1, AtSOC1, and AtFUL were also significantly upregulated in the 35S::MdbHLH48 Arabidopsis lines compared with in the wild-type control (Supplementary Fig. S4E).
为了确定 MdbHLH48 是否影响开花转变，我们在拟南芥 Col-0 中生成了过表达35S::bHLH48-GFP的转基因拟南芥植物。因此，选择具有显着表达的 3 个35S::MdbHLH48系（#1、#3 和 #8）进行进一步分析（图 3G ；补充图 S4D ）。 35S::MdbHLH48品系在长日照（16小时光照/8小时黑暗）条件下表现出明显的早花表型（图3，F和H ）。此外，我们发现AtFT的表达显着升高；相比之下， AtTFL1在35S::MdbHLH48拟南芥幼苗中受到轻微抑制（补充图 S4C ）。与野生型对照相比， 35S::MdbHLH48拟南芥品系中包括AtLFY 、 AtAP1 、 AtSOC1和AtFUL在内的几个开花促进基因的表达水平也显着上调（补充图 S4E ）。

To further confirm the function of MdbHLH48, we also characterized the MdbHLH48 gene by constitutive expression in the tomato cultivar ‘Micro-Tom’. Seven independent lines were obtained, 3 of which were used for monitoring flowering time (Fig. 3, I and J; Supplementary Fig. S4F). The MdbHLH48-overexpressing plants flowered 3 to 5 d earlier than wild-type plants under long-day conditions (Fig. 3K). The tomato flowering-associated genes SlFT, SlLFY, and SlAP1 were promoted, but SlFUL expression did not change in MdbHLH48-overexpressing tomato lines (Supplementary Fig. S4G). Collectively, these results showed that overexpression of MdbHLH48 gene promotes flowering in transgenic Arabidopsis and tomato, suggesting that MdbHLH48 acts as a positive regulator in the floral transition.
为了进一步证实 MdbHLH48 的功能，我们还通过番茄品种“Micro-Tom”中的组成型表达来表征MdbHLH48基因。获得了 7 个独立品系，其中 3 个用于监测开花时间（图 3，I 和 J ；补充图 S4F ）。在长日照条件下， MdbHLH48过表达植物比野生型植物早开花3至5天(图3K )。番茄开花相关基因SlFT 、 SlLFY和SlAP1得到促进，但在MdbHLH48过表达番茄品系中SlFUL表达没有变化（补充图 S4G ）。总的来说，这些结果表明， MdbHLH48基因的过度表达可促进转基因拟南芥和番茄的开花，表明 MdbHLH48 在花转变中充当正调节因子。

We also introduced 35S::MdbHLH48-GFP into apple (GL-3). Based on genomic PCR analysis (Supplementary Fig. S5A), we obtained 2 apple transgenic lines overexpressing MdbHLH48. These 2 lines exhibited higher MdbHLH48 expression levels than the control, as shown by RT-qPCR (Supplementary Fig. S5B). However, these transgenic plants did not display any visible different flowering phenotype compared to the control plants under normal conditions. We speculate that the observation period was not long enough to detect early flowering of 35S::MdbHLH48-GFP transgenic apples, because apple has a long juvenile stage (commonly lasting for 8 yr or more; Foster et al. 2003; Hanke et al. 2007). Notably, MdFT1 and AFL1 were upregulated, but MdTFL1 expression remained unchanged in apices from 1-mo-old transgenic apples compared with in the control (Supplementary Fig. S5C). We predicted that the upregulated MdFT1 may shorten the juvenile period of transgenic apples compared with GL-3 controls. In addition, we observed that the MdbHLH48-overexpressing apple lines exhibited narrower leaves, etiolated leaves, and thinner stems, compared with the GL-3 control (Supplementary Fig. S5D). The specific mechanism of these phenotypes needs to be further studied.
我们还将35S::MdbHLH48-GFP引入苹果 (GL-3)。基于基因组PCR分析（补充图S5A ），我们获得了2个过表达MdbHLH48的苹果转基因品系。如 RT-qPCR 所示（补充图 S5B ），这 2 个系表现出比对照更高的MdbHLH48表达水平。然而，与正常条件下的对照植物相比，这些转基因植物没有表现出任何可见的不同开花表型。我们推测观察期不够长，不足以检测35S::MdbHLH48-GFP转基因苹果的早期开花，因为苹果有一个较长的幼期阶段（通常持续 8 年或更长； Foster 等人，2003 年； Hanke 等人，2003 年）。 2007 ）。值得注意的是，与对照相比，1 个月大转基因苹果的顶端中MdFT1和AFL1表达上调，但MdTFL1表达保持不变（补充图 S5C ）。我们预测，与 GL-3 对照相比，上调的MdFT1可能会缩短转基因苹果的幼年期。此外，我们观察到，与 GL-3 对照相比， MdbHLH48过表达的苹果品系表现出更窄的叶子、黄化的叶子和更细的茎（补充图 S5D ）。这些表型的具体机制有待进一步研究。

MdFT1 and MdTFL1 interact with MdWRKY6
MdFT1 和 MdTFL1 与 MdWRKY6 相互作用

FT and TFL1 have been implicated in transcriptional regulation but do not have DNA-binding domains (Ahn et al. 2006; Corbesier et al. 2007; Hanano and Goto 2011; Goretti et al. 2020); both of them must rely on interactions with other proteins for regulation of relevant gene targets. Studies in several plant species showed that FT and TFL1 function as transcriptional coregulators and can form complexes with the FD protein (Ahn et al. 2006; Corbesier et al. 2007; Zhu et al. 2020). In apple, we previously performed a yeast 2-hybrid (Y2H) library screen using MdTFL1 as a bait protein to identify proteins that potentially interact with MdTFL1 and identified a WRKY protein with a high frequency of occurrence (Zuo et al. 2021b). Phylogenetic analysis showed that this protein is closely related to Arabidopsis AtWRKY6 (Supplementary Fig. S6A) and thus was named MdWRKY6. MdWRKY6 has a single WRKY domain and 1 C2H2 zinc finger motif (Supplementary Fig. S6B) and belongs to group IIb of the WRKY family. Since FT and TFL1 are reported to share similar interacting proteins (Pnueli et al. 2001; Wigge et al. 2005; Zhu et al. 2020; Zuo et al. 2021b), we investigated the physical relationship between MdFT1/MdTFL1 and MdWRKY6 by Y2H assay. The coding sequence (CDS) of MdWRKY6 was inserted into the pGADT7 prey vector and then cotransformed into yeast with the MdFT1 or MdTFL1 fused pGBKT7 bait vector. The results showed that both MdFT1 and MdTFL1 were able to physically interact with MdWRKY6 in yeast cells (Fig. 4A). Analysis using a series of deletion constructs showed that the C-terminal region of MdWRKY6, containing the conserved WRKY domain, is necessary for interaction with MdTFL1 (Supplementary Fig. S7).
FT 和 TFL1 参与转录调控，但不具有 DNA 结合域（ Ahn 等人，2006 年； Corbesier 等人，2007 年； Hanano 和 Goto，2011 年； Goretti 等人，2020 年）；它们都必须依赖于与其他蛋白质的相互作用来调节相关基因靶标。对几种植物物种的研究表明，FT 和 TFL1 作为转录共调节因子发挥作用，并且可以与 FD 蛋白形成复合物（ Ahn 等人，2006 年； Corbesier 等人，2007 年； Zhu 等人，2020 年）。在苹果中，我们之前使用 MdTFL1 作为诱饵蛋白进行了酵母 2-hybrid (Y2H) 文库筛选，以鉴定可能与 MdTFL1 相互作用的蛋白质，并鉴定了出现频率较高的 WRKY 蛋白 ( Zuo et al. 2021b )。系统发育分析表明，该蛋白与拟南芥AtWRKY6密切相关（补充图S6A ），因此被命名为MdWRKY6。 MdWRKY6 具有单个 WRKY 结构域和 1 个 C2H2 锌指基序（补充图 S6B ），属于 WRKY 家族的 IIb 组。由于据报道 FT 和 TFL1 具有相似的相互作用蛋白（ Pnueli et al. 2001 ； Wigge et al. 2005 ； Zhu et al. 2020 ； Zuo et al. 2021b ），我们通过 Y2H 研究了 MdFT1/MdTFL1 和 MdWRKY6 之间的物理关系化验。将MdWRKY6的编码序列(CDS)插入pGADT7猎物载体中，然后与MdFT1或MdTFL1融合的pGBKT7诱饵载体共转化到酵母中。结果表明，MdFT1和MdTFL1均能够与酵母细胞中的MdWRKY6发生物理相互作用(图4A )。 使用一系列缺失构建体进行的分析表明，MdWRKY6 的 C 端区域包含保守的 WRKY 结构域，对于与 MdTFL1 相互作用是必需的（补充图 S7 ）。

We further confirmed the interactions between MdWRKY6 and MdFT1/MdTFL1 using bimolecular fluorescence complementation (BiFC) assays (Fig. 4B). In N. benthamiana leaf epidermal cells coexpressing the C-terminal half of YFP fused to MdWRKY6 (cYFP-MdWRKY6) and the N-terminal half of YFP fused to MdFT1 (nYFP-MdFT1), or N-terminal half of YFP fused to MdTFL1 (nYFP-MdTFL1), strong YFP fluorescence was observed in the nucleus. In contrast, no YFP signal was observed in the negative controls when cYFP-MdWRKY6 and nYFP or cYFP and nYFP-MdFT1/MdTFL1 were cotransformed (Fig. 4B). To further examine these interactions in vivo, we performed a coimmunoprecipitation (Co-IP) assay by transiently expressing MdFT1 or MdTFL1 fused to the GFP tag (MdFT1-GFP/MdTFL1-GFP) and Flag-tagged MdWRKY6 (MdWRKY6-Flag) fusion proteins in N. benthamiana leaves. The MdWRKY6-Flag fusion protein was immunoprecipitated with MdFT1-GFP (Fig. 4C) or MdTFL1-GFP (Fig. 4D), indicating that both MdFT1 and MdTFL1 interact with MdWRKY6 in vivo.
我们使用双分子荧光互补（BiFC）测定进一步证实了MdWRKY6和MdFT1/MdTFL1之间的相互作用（图4B ）。在本塞姆氏烟草叶表皮细胞中，共表达与 MdWRKY6 融合的 YFP C 端一半 (cYFP-MdWRKY6) 和与 MdFT1 融合的 YFP N 端一半 (nYFP-MdFT1)，或与 MdTFL1 融合的 YFP N 端一半(nYFP-MdTFL1)，在细胞核中观察到强YFP荧光。相反，当cYFP-MdWRKY6和nYFP或cYFP和nYFP-MdFT1/MdTFL1共转化时，在阴性对照中没有观察到YFP信号(图4B )。为了进一步检查体内这些相互作用，我们通过瞬时表达与 GFP 标签融合的 MdFT1 或 MdTFL1 (MdFT1-GFP/MdTFL1-GFP) 和带 Flag 标签的 MdWRKY6 (MdWRKY6-Flag) 融合蛋白进行了免疫共沉淀 (Co-IP) 测定。存在于本塞姆氏猪笼草叶中。将MdWRKY6-Flag融合蛋白与MdFT1-GFP(图4C )或MdTFL1-GFP(图4D )进行免疫沉淀，表明MdFT1和MdTFL1两者在体内与MdWRKY6相互作用。

Since MdFT1 and MdTFL1 have an antagonistic role in flowering, interactions of both MdFT1 and MdTFL1 with MdWRKY6 led to the hypothesis that they compete for binding to the target protein. Therefore, to further clarify whether MdFT1 and MdTFL1 could competitively interact with MdWRKY6 protein, a luciferase complementation imaging assay in N. benthamiana leaves was used to examine the interaction intensity of MdTFL1-nLUC/cLUC-MdWRKY6 with or without SK-MdFT1. Coexpression of MdTFL1-nLUC/cLUC-MdWRKY6 elicited robust LUC activity, which was substantially suppressed by the coexpression of SK-MdFT1, and the activity was further reduced with increasing amounts of MdFT1 (Fig. 4E). Evidence of biological replication is provided in Supplementary Fig. S8. These results indicated that MdFT1 weakens the interaction between MdTFL1 and MdWRKY6.
由于 MdFT1 和 MdTFL1 在开花中具有拮抗作用，因此 MdFT1 和 MdTFL1 与 MdWRKY6 的相互作用导致了以下假设：它们竞争与靶蛋白的结合。因此，为了进一步阐明MdFT1和MdTFL1是否可以与MdWRKY6蛋白竞争性相互作用，利用本塞姆氏烟草叶子中的荧光素酶互补成像测定法来检测MdTFL1-nLUC/cLUC-MdWRKY6在有或没有SK-MdFT1的情况下的相互作用强度。 MdTFL1-nLUC/cLUC-MdWRKY6的共表达引发了强大的LUC活性，该活性被SK-MdFT1的共表达显着抑制，并且该活性随着MdFT1量的增加而进一步降低（图4E ）。补充图S8提供了生物复制的证据。这些结果表明MdFT1削弱了MdTFL1和MdWRKY6之间的相互作用。

Subcellular localization analysis showed that the MdWRKY6-GFP fusion protein was localized in the nucleus, in contrast to the diffuse distribution of free GFP (Supplementary Fig. S9A). A trans-acting activity assay in yeast indicated that MdWRKY6 activates the transcription of reporter genes, and its N-terminus (1∼307 amino acids) was essential for this activity (Supplementary Fig. S9B), indicating that MdWRKY6 primarily functions as a transcriptional activator.
亚细胞定位分析表明，MdWRKY6-GFP 融合蛋白定位于细胞核，与游离 GFP 的弥散分布相反（补充图 S9A ）。酵母中的反式作用活性测定表明，MdWRKY6 激活报告基因的转录，其 N 末端（1∼307 个氨基酸）对于该活性至关重要（补充图 S9B ），表明 MdWRKY6 主要作为转录因子发挥作用。激活剂。

Overexpression of MdWRKY6 promotes flowering in Arabidopsis and tomato
MdWRKY6过表达促进拟南芥和番茄开花

Because MdWRKY6 interacts with the key floral proteins MdFT1 and MdTFL1, we hypothesize that MdWRKY6 may be involved in the process of floral transition in apple. To elucidate the expression profile of MdWRKY6, the approximately 2.0 kb promoter region of MdWRKY6 was isolated from ‘Fuji’, fused to the GUS reporter gene, and transformed into Arabidopsis. The pMdWRKY6::GUS construct in transgenic Arabidopsis was expressed in the roots, stems, leaves, and flowers, with particularly high expression in the SAM region (Fig. 5A). In apple, spatial–temporal expression analysis by RT-qPCR showed that MdWRKY6 was highly expressed in the apple spur buds and slightly upregulated with floral transition (Fig. 5, B and C).
由于MdWRKY6与关键的花蛋白MdFT1和MdTFL1相互作用，我们推测MdWRKY6可能参与苹果的花转变过程。为了阐明MdWRKY6的表达谱，从'Fuji'分离MdWRKY6的大约2.0kb启动子区域，与GUS报告基因融合，并转化到拟南芥中。转基因拟南芥中的pMdWRKY6::GUS构建体在根、茎、叶和花中表达，其中在SAM区域中表达特别高(图5A )。在苹果中，RT-qPCR 的时空表达分析表明， MdWRKY6在苹果短枝芽中高表达，并随着花转变而轻微上调（图 5，B 和 C ）。

To further determine whether MdWRKY6 functions in flower transition, we generated MdWRKY6-overexpressing (35S::MdWRKY6-GFP) Arabidopsis plants, and 3 lines (#1, #2, and #4) with especially high MdWRKY6 expression were selected for further analysis (Fig. 5, D and E; Supplementary Fig. S10A). Overexpression of MdWRKY6 greatly reduced the number of rosette leaves compared with that in wild-type seedlings, and flowering occurred significantly earlier in MdWRKY6 overexpression lines than in the wild-type Arabidopsis in long-day conditions (Fig. 5F). The flowering-promoted genes of Arabidopsis, including AtFT, AtLFY, AtAP1, AtSOC1, and AtFUL, were significantly upregulated in the 35S::MdWRKY6 Arabidopsis compared with in the wild-type control (Supplementary Fig. S10B).
为了进一步确定 MdWRKY6 是否在花转变中发挥作用，我们生成了MdWRKY6过表达 ( 35S::MdWRKY6-GFP ) 拟南芥植物，并选择了MdWRKY6表达特别高的 3 个品系（#1、#2 和 #4）进行进一步分析。 （图5、D和E ；补充图S10A ）。与野生型幼苗相比， MdWRKY6的过表达大大减少了莲座叶的数量，并且在长日照条件下， MdWRKY6过表达品系的开花发生时间明显早于野生型拟南芥(图5F )。与野生型对照相比，拟南芥的开花促进基因，包括AtFT 、 AtLFY 、 AtAP1 、 AtSOC1和AtFUL ，在35S::MdWRKY6拟南芥中显着上调（补充图 S10B ）。

We also used 35S::MdWRKY6-GFP for stable transformation in tomato (‘Micro-Tom’) to generate transgenic tomato lines overexpressing MdWRKY6 (Fig. 5H; Supplementary Fig. S10C). We found that the MdWRKY6-overexpressing tomato plants showed early flowering as compared to the wild type (Fig. 5, G and I). The expression of tomato flowering genes SlLFY, SlAP1, and SlFUL were promoted by the ectopic expression of MdWRKY6 in tomato (Supplementary Fig. S10D). Taken together, these results indicated that MdWRKY6 plays a positive role in the control of flowering.
我们还使用35S::MdWRKY6-GFP在番茄（“Micro-Tom”）中进行稳定转化，以生成过表达MdWRKY6的转基因番茄品系（图 5H ；补充图 S10C ）。我们发现，与野生型相比， MdWRKY6过表达的番茄植株显示出较早的开花（图5，G和I ）。番茄中MdWRKY6的异位表达促进了番茄开花基因SlLFY 、 SlAP1和SlFUL的表达（补充图S10D ）。综上所述，这些结果表明MdWRKY6在控制开花中发挥积极作用。

Transcriptional activity of MdWRKY6 is promoted by MdFT1 and repressed by MdTFL1
MdWRKY6 的转录活性由 MdFT1 促进并由 MdTFL1 抑制

In model plants, FT and TFL1 form complexes with the FD and regulate the transition of vegetative to reproductive growth by activating AP1 and LFY (Abe et al. 2005; Hanano and Goto 2011; Kaneko-Suzuki et al. 2018; Zhu et al. 2020). This enables us to investigate the role of FT and TFL1 in modulating MdWRKY6-mediated transcriptional regulation of downstream targets. Previous studies in Arabidopsis showed that several WRKY transcriptional factors control flowering by directly binding to W-box motifs within promoters of floral meristem-identity genes such as LFY and FUL (Li et al. 2016; Yu et al. 2016; Zhang et al. 2018). Also, LFY is the common target gene of FT and TFL1 (Goretti et al. 2020; Zhu et al. 2020). We speculate that MdWRKY6 may recruit MdFT1 and MdTFL1 to the promoter region of apple LFY gene. To investigate this hypothesis, we first investigated the association of MdWRKY6 with the AFL1 promoter (a LFY-like gene of apple) because AFL1 can regulate the floral transition in apple (Wada et al. 2002, 2009). Analysis of AFL1 promoter revealed a putative W-box motif. Y1H assay revealed that MdWRKY6 can directly bind to the AFL1 promoter fragment containing the W-box element (Fig. 6A). Then the EMSA experiment was conducted, and the results confirmed the specificity of the interaction between the MdWRKY6 transcription factor and the W-box region in AFL1 promoter (Fig. 6B).
在模型植物中，FT和TFL1与FD形成复合物，并通过激活AP1和LFY来调节营养生长到生殖生长的转变（ Abe et al. 2005 ； Hanano and Goto 2011 ； Kaneko-Suzuki et al. 2018 ； Zhu et al. 2018）。 2020 ）。这使我们能够研究 FT 和 TFL1 在调节 MdWRKY6 介导的下游靶标转录调控中的作用。先前对拟南芥的研究表明，一些WRKY转录因子通过直接与花分生组织识别基因（如LFY和FUL）启动子内的W-box基序结合来控制开花（ Li et al. 2016 ； Yu et al. 2016 ； Zhang et al. 2016）。 2018 ）。此外， LFY是 FT 和 TFL1 的共同靶基因（ Goretti et al. 2020 ； Zhu et al. 2020 ）。我们推测MdWRKY6可能将MdFT1和MdTFL1招募到苹果LFY基因的启动子区域。为了研究这一假设，我们首先研究了 MdWRKY6 与AFL1启动子（苹果的LFY样基因）的关联，因为 AFL1 可以调节苹果的花转变（ Wada et al. 2002，2009 ） 。对AFL1启动子的分析揭示了一个假定的 W-box 基序。 Y1H测定显示MdWRKY6可以直接结合含有W-box元件的AFL1启动子片段(图6A )。然后进行EMSA实验，结果证实了MdWRKY6转录因子与AFL1启动子中的W-box区域之间相互作用的特异性(图6B )。

To test the function of MdWRKY6 on the regulation of AFL1 in apple, we generated a 35S::MdWRKY6-GFP transgenic ‘Orin’ apple calli (Supplementary Fig. S10E). The expression level of AFL1 was significantly upregulated in the 35S::MdWRKY6-GFP apple calli compared with in the control (empty vector; Fig. 6C), indicating that MdWRKY6 can activate AFL1 transcription.
为了测试 MdWRKY6 对苹果AFL1调节的功能，我们生成了35S::MdWRKY6-GFP转基因“Orin”苹果愈伤组织（补充图 S10E ）。与对照（空载体；图6C ）相比，35S::MdWRKY6-GFP苹果愈伤组织中AFL1的表达水平显着上调，表明MdWRKY6可以激活AFL1转录。

To further analyze the effects of MdFT1 and MdTFL1 on the transcriptional activation activity of MdWRKY6, a transient expression assay in N. benthamiana leaves was performed. The AFL1 promoter (2.0 kb) was fused with LUC as the reporter, and the highly expressed MdWRKY6, MdFT1, or MdTFL1 was used as the effector (Fig. 6D). The results showed that MdWRKY6 alone stimulated the expression of AFL1 reporter (Fig. 6E), consistent with its role as a transcriptional activator (Supplementary Fig. S9B). Cotransfection of MdWRKY6 and MdFT1 plasmids was able to produce substantially higher pAFL1-LUC activity, while cotransfection of MdTFL1 and MdWRKY6 reduced the pAFL1-LUC activity compared with MdWRKY6 alone. However, the expression of MdFT1 or MdTFL1 alone with pAFL1-LUC had no effect on AFL1 promoter activity (Fig. 6E). In addition, notably, the activation of AFL1 by coexpression of MdFT1 and MdWRKY6 was significantly reduced when MdTFL1 was copresent (Fig. 6F), indicating MdTFL1 and MdFT1 may play an antagonistic role by interacting with MdWRKY6. Collectively, these results demonstrate that MdFT1 may act as a transcriptional coactivator to enhance the transcriptional activation of AFL1 by MdWRKY6, whereas MdTFL1 negatively modulates transcriptional activation of AFL1 by MdWRKY6. The differential regulation of MdTFL1 and MdFT1 on MdWRKY6-mediated transcriptional activation may be through competitive interaction with MdWRKY6.
为了进一步分析 MdFT1 和 MdTFL1 对 MdWRKY6 转录激活活性的影响，在本塞姆氏烟草叶子中进行了瞬时表达测定。 AFL1启动子(2.0kb)与作为报告基因的LUC融合，并且使用高表达的MdWRKY6 、 MdFT1或MdTFL1作为效应子(图6D )。结果表明，MdWRKY6 单独刺激AFL1报告基因的表达（图 6E ），与其作为转录激活剂的作用一致（补充图 S9B ）。 MdWRKY6和MdFT1质粒的共转染能够产生显着更高的pAFL1- LUC活性，而与单独的MdWRKY6相比， MdTFL1和MdWRKY6的共转染降低了pAFL1- LUC活性。然而， MdFT1或MdTFL1单独与pAFL1- LUC的表达对AFL1启动子活性没有影响(图6E )。此外，值得注意的是，当MdTFL1共存时， MdFT1和MdWRKY6共表达对AFL1的激活显着降低(图6F )，表明MdTFL1和MdFT1可能通过与MdWRKY6相互作用发挥拮抗作用。总的来说，这些结果表明，MdFT1 可能充当转录共激活因子，增强 MdWRKY6 对AFL1的转录激活，而 MdTFL1 负向调节 MdWRKY6 对AFL1的转录激活。 MdTFL1 和 MdFT1 对 MdWRKY6 介导的转录激活的差异调节可能是通过与 MdWRKY6 的竞争性相互作用实现的。

Discussion 讨论

Apple flower bud formation is the key prerequisite for normal flowering and fruit. Some apple varieties, such as ‘Fuji’ and ‘Honeycrisp’, have an unstable flowering rate, which is closely related to the flower bud formation in early summer. Therefore, understanding the molecular mechanisms of flower bud formation and the gene regulatory network is consequently important for apple production. FT1 and TFL1 have opposite functions in regulating plant flowering, and the balance of their ratios plays central roles in determining the fate of bud meristem as vegetative or reproductive and has a considerable impact on plant architecture and yield (Mimida et al. 2013; Higuchi 2018; Gaston et al. 2021). In this study, we identified a MdbHLH48 transcriptional factor as a flowering promotor that could differentially regulate the transcriptional levels of MdFT1 and MdTFL1. MdFT1 and MdTFL1 as transcriptional cofactors further interact with MdWRKY6 to regulate the expression of the downstream flowering gene AFL1 and thus mediate flowering transition.
苹果花芽的形成是苹果正常开花结果的关键前提。一些苹果品种，如‘Fuji’和‘Honeycrisp’，开花率不稳定，这与初夏花芽的形成密切相关。因此，了解花芽形成的分子机制和基因调控网络对于苹果生产具有重要意义。 FT1和TFL1在调节植物开花方面具有相反的功能，其比例的平衡在决定芽分生组织营养或生殖的命运方面起着核心作用，并对植物结构和产量有相当大的影响（ Mimida等人，2013年； Higuchi，2018年）加斯顿等人，2021 ）。在这项研究中，我们鉴定了 MdbHLH48 转录因子作为开花启动子，可以差异调节MdFT1和MdTFL1的转录水平。 MdFT1和MdTFL1作为转录辅助因子进一步与MdWRKY6相互作用，调节下游开花基因AFL1的表达，从而介导开花转变。

In apple, the expression of MdFTs and MdTFL1s and the function of their products in regulating flowering have been well studied (Kotoda et al. 2006; Mimida et al. 2009; Kotoda et al. 2010; Tränkner et al. 2010; Mimida et al. 2011a, 2011b; Flachowsky et al. 2012). The expression patterns of MdFT1 and MdFT2 were different in that MdFT1 was expressed mainly in the apical buds and increased concurrently with floral induction in the apical buds, while MdFT2 was mainly expressed in stems and young fruits (Fig. 1; Kotoda et al. 2010). Thus, 2 FT-like proteins may undergo functional divergence in apple. Considering that MdFT1 but not MdFT2 was highly expressed in apical buds and that it was expressed more higher in spur buds than lateral buds during the flower induction stage (Fig. 1), MdFT1 plays a key role in the transition to flowering in apple. Indeed, the transgenic apple overexpressing MdFT1 showed an extreme early-flowering phenotype (Kotoda et al. 2010; Tränkner et al. 2010). Notably, in several species such as A. thaliana, FT is highly expressed in leaves, and the protein is transported to SAM through the phloem for its function. In our study, we found that MdFT1 is mainly highly expressed in the apical buds but relatively low in the leaves, which is consistent with the previous findings (Kotoda et al. 2010; Haberman et al. 2016). Likewise, in Japanese pear (Pyrus pyrifolia Nakai), which also belongs to the family Rosaceae and is genetically close to apple, PpFT1 induction was observed during the early stage of floral transition in buds but not in leaves (Bai et al. 2017). These results suggest that unlike the induced FT expression in leaves observed in Arabidopsis, in apple, SAM may receive floral signals that directly induce apple MdFT1 expression in the shoot apex and thereby induce flowering.
在苹果中， MdFTs和MdTFL1s的表达及其产物在调节开花中的功能已得到充分研究( Kotoda et al. 2006 ; Mimida et al. 2009 ; Kotoda et al. 2010 ; Tränkner et al. 2010 ; Mimida et al. 2011a ， 2011b ；弗拉乔夫斯基等人。 MdFT1和MdFT2的表达模式不同， MdFT1主要在顶芽中表达，并在顶芽中与成花诱导同时增加，而MdFT2主要在茎和幼果中表达（图1 ； Kotoda等，2010） ）。因此，2 个 FT 样蛋白在苹果中可能会出现功能分歧。考虑到在花诱导阶段， MdFT1而不是MdFT2在顶芽中高表达，并且在短枝芽中表达高于侧芽(图1 )，因此MdFT1在苹果向开花的过渡中起着关键作用。事实上，过表达MdFT1的转基因苹果表现出极端的早花表型（ Kotoda 等人，2010 年； Tränkner 等人，2010 年）。值得注意的是，在拟南芥等一些物种中， FT在叶子中高度表达，并且该蛋白质通过韧皮部转运至 SAM 发挥其功能。在我们的研究中，我们发现MdFT1主要在顶芽中高表达，但在叶片中表达量相对较低，这与之前的研究结果一致( Kotoda et al. 2010 ; Haberman et al. 2016 )。 同样，在同样属于蔷薇科且在遗传上与苹果接近的日本梨（ PyruspyrifoliaNakai ）中，在花芽转变的早期阶段观察到PpFT1诱导，但在叶子中却没有观察到（ Bai 等人，2017 ）。这些结果表明，与在拟南芥中观察到的叶片中诱导的FT表达不同，在苹果中，SAM 可能接收花信号，直接诱导苹果茎尖的MdFT1表达，从而诱导开花。

Unlike MdFT1, the expression of 2 apple TFL1-like genes declined sharply during floral induction (Fig. 1D). The strong decrease in expression of these 2 TFL1 homologs during the anticipated period for floral induction has also been reported previously (Kotoda and Wada 2005; Hättasch et al. 2008; Mimida et al. 2009; Guitton et al. 2016; Haberman et al. 2016; Zhang et al. 2019; Gottschalk et al. 2021), implying that the reduction of the MdTFL1/MdTFL1a expression appears to be necessary for apple floral initiation. In addition, under the action of flowering inhibitory signals such as heavy fruit load and gibberellin, the expression of apple TFL1-like gene was upregulated (Haberman et al. 2016; Zhang et al. 2019; Gottschalk et al. 2021; Zuo et al. 2021b), consistent with the role of its product in inhibiting apple flower bud formation (Kotoda et al. 2006; Flachowsky et al. 2012). Taken together, it seems that the precise regulation of MdTFL1 and MdFT1 expression is very important for flowering in apple, with high MdFT1 expression favoring flower bud formation while high MdTFL1 expression inhibiting flower bud formation.
与MdFT1不同，2个苹果TFL1样基因的表达在花诱导过程中急剧下降（图1D ）。先前也报道过这 2 个TFL1同源物在花诱导期的预期表达量大幅下降（ Kotoda 和 Wada 2005 ； Hättasch 等人 2008 ； Mimida 等人 2009 ； Guitton 等人 2016 ； Haberman 等人 2016）。 2016 ； Zhang 等人 2019 ； Gottschalk 等人 2021 ），这表明MdTFL1/MdTFL1a表达的减少似乎对于苹果花的形成是必要的。此外，在重果负荷、赤霉素等开花抑制信号的作用下，苹果TFL1样基因的表达上调( Haberman et al. 2016 ； Zhang et al. 2019 ； Gottschalk et al. 2021 ； Zuo et al. .2021b ），与其产品抑制苹果花芽形成的作用一致（ Kotoda 等人，2006 年； Flachowsky 等人，2012 年）。综上所述， MdTFL1和MdFT1表达的精确调控对于苹果开花非常重要， MdFT1高表达有利于花芽形成，而MdTFL1高表达则抑制花芽形成。

So how is the expression of FT and TFL1 precisely regulated? In Arabidopsis, transcription of FT is a point of convergence of different seasonal cues and is regulated by multiple transcription factors from different signal pathways. For example, CONSTANCS (CO) activates FT transcription in leaves under long days in the photoperiodic flowering pathway (Samach et al. 2000). Two bHLH transcription factors, bHLH48 and bHLH60, control GA-mediated flowering by directly upregulating the expression of the FT gene (Li et al. 2017). A recent study in citrus found that CiNF-YA1 (nuclear factor YA) activates CiFT expression to control flowering in response to drought and low temperatures (Zhou et al. 2022). However, the signals and factors that regulate TFL1 expression have not yet been established. In this study, somewhat surprisingly, we observed that the transcripts of both MdFT1 and MdTFL1 are regulated by a common transcription factor, MdbHLH48, which can directly bind to their promoter regions containing E-box elements. MdbHLH48 belongs to bHLH subgroup XII, and CIBs in this subgroup are also found to be involved in regulating flower transition in Arabidopsis (Liu et al. 2013). In this work, in particular, we found that MdbHLH48 promotes flowering by activating the expression of MdFT1, on the one hand, and by repressing the expression of MdTFL1, on the other hand (Fig. 2). This “double insurance” is more conducive to the smooth induction of apple flower buds and thus produces a sufficient number of flower buds in apple. Such bifunctional regulation by a single transcription factor (where one gene is positively regulated, and another is negatively regulated) may be widespread. For example, SPL10 regulates age-mediated flowering in Arabidopsis by positively inducing WRKY12 expression and negatively inducing WRKY13 expression (Ma et al. 2020). Arabidopsis WUSCHEL acts as a repressor in the regulation of floral meristem size and as an activator in the regulation of AG expression (Ikeda et al. 2009). Further studies are needed to illustrate how transcription factors bifunctionally control other physiological processes. Whether a similar MdbHLH48-MdFT1/MdTFL1 regulatory module exists in other plants (e.g. the model plant Arabidopsis) needs further investigation.
那么FT和TFL1的表达是如何精准调控的呢？在拟南芥中， FT转录是不同季节线索的汇聚点，并受到来自不同信号通路的多种转录因子的调节。例如，CONSTANCS (CO) 在光周期开花途径中的长日照下激活叶子中的FT转录（ Samach 等人，2000 ）。两种bHLH转录因子bHLH48和bHLH60通过直接上调FT基因的表达来控制GA介导的开花（ Li et al. 2017 ）。最近的一项柑橘研究发现，CiNF-YA1（核因子 YA）可激活CiFT表达，以响应干旱和低温来控制开花（ Zhou et al. 2022 ）。然而，调节TFL1表达的信号和因子尚未确定。在这项研究中，有些令人惊讶的是，我们观察到MdFT1和MdTFL1的转录本均受到共同转录因子 MdbHLH48 的调节，该转录因子可以直接与其含有 E-box 元件的启动子区域结合。 MdbHLH48属于bHLH XII亚组，该亚组中的CIB也被发现参与调节拟南芥花的转变( Liu et al. 2013 )。特别是在本工作中，我们发现MdbHLH48一方面通过激活MdFT1的表达，另一方面通过抑制MdTFL1的表达来促进开花(图2 )。这种“双保险”更有利于苹果花芽的顺利诱导，从而使苹果产生足够数量的花芽。 这种由单一转录因子进行的双功能调节（其中一个基因受到正向调节，而另一个基因受到负向调节）可能很普遍。例如，SPL10 通过正向诱导WRKY12表达和负向诱导WRKY13表达来调节拟南芥中年龄介导的开花（ Ma et al. 2020 ）。拟南芥 WUSCHEL 在花分生组织大小的调节中充当抑制子，在AG表达的调节中充当激活子（ Ikeda 等人，2009 ）。需要进一步的研究来说明转录因子如何双功能地控制其他生理过程。其他植物（如模式植物拟南芥）中是否存在类似的MdbHLH48- MdFT1 / MdTFL1调控模块还需要进一步研究。

Many bHLH transcription factors have been reported to regulate plant flowering (Liu et al. 2013; Ye et al. 2023; Wang et al. 2023b). For example, in citrus, CibHLH96 participates in the regulation of drought-induced flowering by activating the expression of CiFD (Ye et al. 2023). Apple MdbHLH4 promotes the transition to flowering by interacting with FLOWERING LOCUS C (FLC) and activating the transcription of FT (Wang et al. 2023b). Here, we found that MdbHLH48 is mainly expressed in apical buds and gradually accumulated during the flowering induction period. Overexpression of MdbHLH48 in Arabidopsis and tomato clearly promoted flowering (Fig. 3), similar to overexpression of the Arabidopsis bHLH48 gene (Li et al. 2017). However, early flowering in apple overexpressing MdbHLH48 cannot be observed in a short time, due to the relatively long juvenile period of apple, which commonly lasts for 4 to 8 yr or more (Foster et al. 2003; Hanke et al. 2007). Although MdFT1 is also significantly upregulated in 35S::MdbHLH48-GFP transgenic apples, it is hypothesized that the accumulation of MdFT1 may not reach the necessary threshold for apple flowering, thereby limiting transgenic plants to flower within a short period of time. A similar phenomenon has been observed in some woody fruit trees transformed with other flowering genes. For example, overexpression of the Arabidopsis floral promoter gene LFY in apple resulted in a columnar phenotype with reduced internode length but did not expedite the flowering process (Flachowsky et al. 2010). Similarly, overexpression of the citrus flowering gene CiMADS43 in citrus did not induce an early flowering phenotype, despite its extremely early flowering characteristics in transgenic Arabidopsis (Ye et al. 2021). Based on the above, therefore, the contribution of MdbHLH48 to apple flowering and the relationship between MdbHLH48 and MdFT1/MdTFL1 need to be further evaluated in future work in apple.
据报道，许多 bHLH 转录因子可调节植物开花（ Liu et al. 2013 ； Ye et al. 2023 ； Wang et al. 2023b ）。例如，在柑橘中，CibHLH96 通过激活CiFD的表达参与干旱诱导开花的调节（ Ye et al. 2023 ）。苹果 MdbHLH4 通过与 FLOWERING LOCUS C (FLC) 相互作用并激活FT转录来促进开花过渡 ( Wang et al. 2023b )。在这里，我们发现MdbHLH48主要在顶芽中表达，并在开花诱导期逐渐积累。 MdbHLH48在拟南芥和番茄中的过表达明显促进开花（图3 ），类似于拟南芥bHLH48基因的过表达（ Li等人，2017 ）。然而，过表达MdbHLH48的苹果不能在短时间内观察到早期开花，因为苹果的幼年期相对较长，通常持续4至8年或更长时间（ Foster等人，2003 ； Hanke等人，2007 ）。尽管MdFT1在35S::MdbHLH48-GFP转基因苹果中也显着上调，但推测MdFT1的积累可能达不到苹果开花所需的阈值，从而限制了转基因植物在短时间内开花。在一些用其他开花基因转化的木本果树中也观察到了类似的现象。 例如，拟南芥花启动子基因LFY在苹果中的过度表达导致节间长度缩短的柱状表型，但并没有加速开花过程（ Flachowsky 等人，2010 ）。同样，柑橘开花基因CiMADS43在柑橘中的过度表达并没有诱导早期开花表型，尽管其在转基因拟南芥中具有极早开花的特征（ Ye et al. 2021 ）。因此，基于上述，MdbHLH48对苹果开花的贡献以及MdbHLH48与MdFT1 / MdTFL1之间的关系需要在未来的苹果工作中进一步评估。

Because FT and TFL1 lack predicted DNA-binding domains, FT and TFL1 functions must rely on interactions with other proteins for regulation of relevant related target genes (Hanano and Goto 2011). An existing model proposes that TFL1 or FT forms a complex with the FD in SAM, and the complex controls flowering by regulating the expression of AP1 and LFY (Abe et al. 2005; Wigge et al. 2005; Hanano and Goto 2011; Randoux et al. 2014; Kaneko-Suzuki et al. 2018; Collani et al. 2019; Zhu et al. 2020). Other transcription factors may also be involved in the complex containing FT or TFL1 to participate in the regulation of flowering. Previously, we identified several MdTFL1-interacting proteins (including MdNF-YC2, MdTCP14, and Md5PTase2) that also interact with MdFT1 protein (Zuo et al. 2021b), suggesting that MdTFL1 and MdFT1 share the same partners, as observed in other studies (Pnueli et al. 2001; Wigge et al. 2005; Zhu et al. 2020; Zuo et al. 2021b). In this study, we found that both MdTFL1 and MdFT1 can strongly interact with MdWRKY6 transcription factor, and the interaction between MdTFL1 and MdWRKY6 is weaker when MdFT1 is present (Fig. 4), suggesting MdTFL1 and MdFT1 compete for binding to MdWRKY6.
由于 FT 和 TFL1 缺乏预测的 DNA 结合域，FT 和 TFL1 的功能必须依赖于与其他蛋白质的相互作用来调节相关的相关靶基因 ( Hanano 和 Goto 2011 )。现有模型提出TFL1或FT与SAM中的FD形成复合物，该复合物通过调节AP1和LFY的表达来控制开花( Abe et al. 2005 ; Wigge et al. 2005 ; Hanano and Goto 2011 ; Randoux et al. 2014 年；Kaneko - Suzuki 等人，2019 年；其他转录因子也可能参与含有FT或TFL1的复合物参与开花的调控。此前，我们鉴定了几种与 MdTFL1 相互作用的蛋白（包括 MdNF-YC2、MdTCP14 和 Md5PTase2），它们也与 MdFT1 蛋白相互作用（ Zuo et al. 2021b ），这表明 MdTFL1 和 MdFT1 具有相同的伙伴，正如在其他研究中观察到的那样（ Pnueli等人，2001 年； Zuo 等人，2021 年。在本研究中，我们发现MdTFL1和MdFT1都可以与MdWRKY6转录因子产生强烈的相互作用，并且当MdFT1存在时，MdTFL1和MdWRKY6之间的相互作用较弱（图4 ），表明MdTFL1和MdFT1竞争与MdWRKY6的结合。

The role of WRKY transcription factors in regulating flowering has been documented (Li et al. 2016; Yu et al. 2016; Zhang et al. 2018). In this work, we showed that MdWRKY6 helps to promote flowering by binding directly to the AFL1 promoter and activating AFL1 expression (Fig. 6). It was more recently demonstrated in Arabidopsis that the master regulator of floral fate, LFY, is a target under dual opposite regulation by TFL1 and FT (Zhu et al. 2020). In apple, AFL1 (an apple LFY-like gene) is expressed in the floral buds, and its product regulates the floral transition (Wada et al. 2002, 2009), and AFL1/AFL2 are upregulated in MdTFL1-silenced and MdFT1-overexpressing transgenic apples (Kotoda et al. 2006, 2010), indicating that AFL1 probably functions downstream of MdTFL1 and MdFT1. Consistent with these findings, in this study, we found that MdTFL1 and MdFT1 exert opposite regulatory roles, negatively and positively regulating MdWRKY6-mediated transcription of target gene AFL1, respectively, and that MdTFL1 could substantially interfere with the transcriptional activation of AFL1 by MdFT1-MdWRKY6. We proposed that, similar to the finding previously (Abe et al. 2005; Wigge et al. 2005; Ahn et al. 2006; Hanano and Goto 2011; Kaneko-Suzuki et al. 2018; Zhu et al. 2020), MdFT1 and MdTFL1 interact with MdWRKY6 to form florigen activation complex or florigen inhibition complex, respectively, and the balance of MdFT1-MdWRKY6 and MdTFL1-MdWRKY6 complexes plays a crucial role in fine-tuning apple flower bud formation.
WRKY 转录因子在调节开花中的作用已被记录（ Li et al. 2016 ； Yu et al. 2016 ； Zhang et al. 2018 ）。在这项工作中，我们表明MdWRKY6通过直接结合AFL1启动子并激活AFL1表达来帮助促进开花（图6 ）。最近在拟南芥中证明，花命运的主要调节因子LFY是 TFL1 和 FT 双重相反调节的目标（ Zhu et al. 2020 ）。在苹果中， AFL1 （苹果LFY样基因）在花蕾中表达，其产物调节花的转变（ Wada et al. 2002，2009 ） ，并且AFL1 / AFL2在MdTFL1沉默和MdFT1过表达中上调转基因苹果 ( Kotoda et al. 2006 , 2010 )，表明 AFL1 可能在 MdTFL1 和 MdFT1 的下游发挥作用。与这些发现一致，在本研究中，我们发现MdTFL1和MdFT1发挥相反的调节作用，分别负向和正向调节MdWRKY6介导的靶基因AFL1的转录，并且MdTFL1可以显着干扰MdFT1对AFL1的转录激活。 MdWRKY6。我们提出，与之前的发现类似（ Abe et al. 2005 ； Wigge et al. 2005 ； Ahn et al. 2006 ； Hanano and Goto 2011 ； Kaneko-Suzuki et al. 2018 ； Zhu et al. 2018）。 2020），MdFT1和MdTFL1与MdWRKY6相互作用，分别形成成花素激活复合物或成花素抑制复合物，并且MdFT1-MdWRKY6和MdTFL1-MdWRKY6复合物的平衡在微调苹果花芽形成中起着至关重要的作用。

In summary, based on the findings of this study and previous reports, we deduced a possible model for the antagonistic regulation of apple flower induction by MdTFL1 and MdFT1 (Fig. 7): During flower induction stages, the floral activation signal is transmitted to the key transcription factor MdbHLH48, which activates MdFT1 while repressing MdTFL1 transcription. The abundant MdFT1 protein then interacts with MdWRKY6 to facilitate phase transition to flowering through activation of AFL1 in apple. In contrast, MdTFL1 competes with MdFT1 for binding to MdWRKY6 to perform the opposite role, which is to negatively regulate AFL1 expression.
综上所述，基于本研究的结果和之前的报道，我们推导出了MdTFL1和MdFT1对苹果花诱导的拮抗调节的可能模型（图7 ）：在花诱导阶段，花激活信号传递到关键转录因子 MdbHLH48，可激活MdFT1，同时抑制MdTFL1转录。然后丰富的 MdFT1 蛋白与 MdWRKY6 相互作用，通过激活苹果中的AFL1促进向开花的相变。相反，MdTFL1 与 MdFT1 竞争与 MdWRKY6 的结合，从而发挥相反的作用，即负向调节AFL1 的表达。

Materials and methods 材料和方法

Plant materials and growth conditions
植物材料和生长条件

Plant materials were sampled from 6-yr-old ‘Fuji’ apple (Malus domestica Borkh.) trees with normal fruit bearing, which were grown in the orchard under natural conditions at Northwest A&F University (Qianyang, Shaanxi Province, China). To investigate the tissue-specific expression, tissues of roots, phloem of stems (about 10 cm from top of the branch), mature leaves, apical buds of short shoots at the flower induction stage, flowers, and young fruit were collected during the spring and summer seasons of 2020. To detect gene expression patterns, the apical buds of short shoots (also called spur buds) and lateral buds of 1-yr shoots were collected at 15, 30, 45, 50, 60, and 70 DAFB in spring 2021, which covered the period of floral induction and floral initiation (Foster et al. 2003). There were 3 biological replicates, and each was mixed from 3 trees with similar bloom density (60% to 70%). For RNA-seq analysis, the samples of the apical buds from ‘Fuji’ during flower induction (15, 30, and 45 DAFB) were collected and used for RNA-seq. Detailed data analyses are provided in the Supplementary Figures and Materials and Methods.
植物材料采样自西北农林科技大学（中国陕西省黔阳市）果园自然条件下生长的6年生、正常结果的“富士”苹果树。为了研究组织特异性表达，春季收集了根部组织、茎韧皮部组织（距离枝条顶部约10cm）、成熟叶、花诱导期短枝顶芽、花和幼果。 2020年和夏季。为了检测基因表达模式，在春季15、30、45、50、60和70 DAFB收集短芽的顶芽（也称为短芽）和1年芽的侧芽2021 年，涵盖花诱导期和花起始期（ Foster et al. 2003 ）。有 3 个生物重复，每个重复由 3 棵具有相似开花密度（60% 至 70%）的树混合而成。对于 RNA-seq 分析，收集了花诱导期间“Fuji”顶芽的样本（15、30 和 45 DAFB）并用于 RNA-seq。补充数据和材料和方法中提供了详细的数据分析。

The ‘Orin’ apple calli were grown on MS medium (with 3% [w/v] sucrose) with 1.5 mg/L 2-4-D and 1.5 mg/L 6-BA and were kept in darkness at 25 °C. The apple calli were subcultured every half-month before being used for genetic transformation. Arabidopsis (A. thaliana) ecotype Col-0 and tomato (S. lycopersicum) cv. ‘Micro-Tom’ were used as the background plants for genetic transformation. N. benthamiana plants were used for the transient expression assays. Arabidopsis seeds were surface sterilized with 10% hypochlorite and sown on 0.5× MS solid medium and incubated at 4 °C for 3 d to synchronize germination before being transplanted to soil composed of peat, vermiculite, and perlite (2:1:1 ratio). Subsequently, the seedlings were transplanted to soil (peat:vermiculite:perlite, 2:1:1). Tomato seeds were sown in the same soil mix. N. benthamiana seeds were initially germinated on moist filter paper before transferring the seedling to the soil. All those plant seedlings were grown in growth chambers under long-day conditions with a 16-h light/8-h dark cycle at temperatures of 22 °C for Arabidopsis and 25 °C for tomato and N. benthamiana.
“Orin”苹果愈伤组织在含有 1.5 mg/L 2-4-D 和 1.5 mg/L 6-BA 的 MS 培养基（含 3% [w/v] 蔗糖）上生长，并在 25 °C 的黑暗条件下保存。苹果愈伤组织每半个月传代一次，然后用于遗传转化。拟南芥 ( A. thaliana ) 生态型 Col-0 和番茄 ( S. lycopersicum ) cv. “Micro-Tom”被用作遗传转化的背景植物。本塞姆氏烟草植物用于瞬时表达测定。拟南芥种子用10%次氯酸盐进行表面消毒，播种在0.5×MS固体培养基上，在4℃下培养3天以同步发芽，然后移植到由泥炭、蛭石和珍珠岩（2:1:1比例）组成的土壤中。随后，将幼苗移植到土壤中（泥炭：蛭石：珍珠岩，2：1：1）。将番茄种子播种在相同的土壤混合物中。本塞姆氏烟草种子最初在潮湿的滤纸上发芽，然后将幼苗转移到土壤中。所有这些植物幼苗均在长日照条件下生长，其中拟南芥温度为 22°C，番茄和本塞姆氏烟草温度为 25°C，光照周期为 16 小时/黑暗周期为 8 小时。

RNA isolation and expression analysis by RT-qPCR
RNA 分离和 RT-qPCR 表达分析

Total RNA was isolated from Arabidopsis and tomato seedlings using TRIzol reagent (Thermo Fisher Scientific) and from previously frozen apple tissues with a CTAB-based method. Reverse transcription was conducted using 1 µg of total RNA from each sample with PrimeScript RT reagent kit (TaKaRa, Dalian, China). RT-qPCR assay was performed as described previously (Zuo et al. 2021a). The AtTUB2 (AT5G62690) for transgenic Arabidopsis, UBI (Solyc01g056940) for transgenic tomato, and HistoneH3 (LOC103406086; Kotoda et al. 2010) for apple tissues were used as reference. Three biological replicates were used for each sample. Data shown in the RT-qPCR data represent the means ± Se of 3 replicate PCR reactions.
使用 TRIzol 试剂 (Thermo Fisher Scientific) 从拟南芥和番茄幼苗中分离总 RNA，并使用基于 CTAB 的方法从先前冷冻的苹果组织中分离总 RNA。使用PrimeScript RT试剂盒（TaKaRa，大连，中国）使用来自每个样品的1μg总RNA进行逆转录。 RT-qPCR 测定如前所述进行（ Zuo 等人，2021a ）。使用用于转基因拟南芥的AtTUB2 (AT5G62690)、用于转基因番茄的UBI (Solyc01g056940)和用于苹果组织的HistoneH3 (LOC103406086； Kotoda等人2010 )作为参考。每个样品使用三个生物复制品。 RT-qPCR 数据中显示的数据代表 3 次重复 PCR 反应的平均值 ± Se。

Tissue sections 组织切片

Buds were collected and fixed in FAA buffer (3.7% [v/v] formaldehyde, 5% [v/v] acetic acid, and 50% [v/v] ethanol) and then embedded in paraffin. Specimens were cut into 12-mm-thick longitudinal sections with a microtome (LEICA RM2016) and stained with toluidine blue. The sections were observed under a light microscope (Nikon ECLIPSE E100, Japan).
收集芽并固定在 FAA 缓冲液（3.7% [v/v] 甲醛、5% [v/v] 乙酸和 50% [v/v] 乙醇）中，然后包埋在石蜡中。用切片机（LEICA RM2016）将样本切成12毫米厚的纵向切片并用甲苯胺蓝染色。在光学显微镜（Nikon ECLIPSE E100，日本）下观察切片。

Plant transformation 植物转化

To generate the overexpressing transgenic plants, the full-length CDS of MdbHLH48 or MdWRKY6 were amplified from ‘Nagafu No. 2' (a Fuji variety) and inserted into the pCAMBIA2300-GFP vector that was driven by the CaMV 35S promoter to form overexpression constructs 35S::MdbHLH48-GFP and 35S::MdWRKY6-GFP. To construct pMdWRKY6::GUS, a region of approximately 2.0 kb of ATG upstream of MdWRKY6 was cloned into the vector pCAMBIA1381-GUS. Details of the primers used for constructing vectors are listed in Supplementary Table S2.
为了生成过表达转基因植物，从“Nagafu No. 2”（富士品种）扩增MdbHLH48或MdWRKY6的全长 CDS，并将其插入由CaMV 35S启动子驱动的 pCAMBIA2300-GFP 载体中，以形成过表达构建体35S::MdbHLH48-GFP和35S::MdWRKY6-GFP 。为了构建pMdWRKY6::GUS ，将MdWRKY6上游大约2.0kb的ATG区域克隆到载体pCAMBIA1381-GUS中。用于构建载体的引物的详细信息列于补充表S2中。

For A. thaliana transformation, the 3 recombinant plasmids described above were introduced into the Col-0 ecotype via the Agrobacterium tumefaciens GV3101-mediated floral dip method. Seeds of the transgenic plants were individually harvested and screened for corresponding vector resistance. T3 homozygous transgenic lines were used for further phenotypic analysis. Flowering time was measured as days from seeding to flowering.
对于拟南芥转化，通过根癌农杆菌GV3101 介导的浸花法将上述 3 个重组质粒引入 Col-0 生态型。单独收获转基因植物的种子并筛选相应的载体抗性。 T3纯合转基因系用于进一步的表型分析。开花时间以从播种到开花的天数来测量。

For tomato (S. lycopersicum) transformation, the 35S::MdbHLH48-GFP and 35S::MdWRKY6-GFP constructs inserted into A. tumefaciens strain EHA105 cells were stably transformed to tomato (‘Micro-Tom’) using the leaf disk method. The transformants were selected on half-strength MS agar plates containing 50 mg/L kanamycin, and the transgenic lines were checked by PCR for the presence of the construct and by RT-qPCR for its expression.
对于番茄（ S. lycopersicum ）转化，使用叶盘法将插入根癌农杆菌菌株EHA105细胞中的35S::MdbHLH48-GFP和35S::MdWRKY6-GFP构建体稳定转化至番茄（“Micro-Tom”）。在含有 50 mg/L 卡那霉素的半强度 MS 琼脂平板上选择转化体，并通过 PCR 检查构建体的存在并通过 RT-qPCR 检查其表达。

For apple calli transformation, the 35S::MdbHLH48-GFP, 35S::MdWRKY6-GFP, and 35S::GFP were introduced into the ‘Orin’ apple calli via the A. tumefaciens EHA105-mediated method described by Wang et al. (2018). Resistant calli showing stable growth were used for further investigation. The integration of the transgene into the different transgenic lines was confirmed by PCR amplification.
对于苹果愈伤组织转化，通过Wang 等人描述的根癌农杆菌EHA105 介导的方法将35S::MdbHLH48-GFP 、 35S::MdWRKY6-GFP和35S::GFP引入“Orin”苹果愈伤组织。 （2018） 。显示稳定生长的抗性愈伤组织用于进一步研究。通过PCR扩增证实转基因整合到不同的转基因系中。

Sequence alignments and phylogenetic analysis
序列比对和系统发育分析

Amino acid sequences were aligned using ClustalW algorithm. Phylogenetic trees were then constructed with MEGA 7.0 software using the neighbor-joining (NJ) method. These parameters were used in the NJ method: bootstrap (1,000 replicates), complete deletion, and amino: p distance.
使用 ClustalW 算法比对氨基酸序列。然后使用 MEGA 7.0 软件使用邻接（NJ）方法构建系统发育树。 NJ 方法中使用了这些参数：bootstrap（1,000 次重复）、完全删除和氨基：p 距离。

Y1H assay Y1H检测

To test the binding of MdbHLH48 to MdTFL1/MdFT1 promoters and of MdWRKY6 to AFL1 promoter in yeast, Y1H assays were performed using a Matchmaker Gold Yeast One-Hybrid System Kit (Clontech). The MdTFL1 and MdFT1 promoter fragments containing the bHLH-binding core sequence (GTAC) and the AFL1 promoter fragment containing W-box elements were independently cloned into the pAbAi vector, respectively. The recombinant promoter-AbAi plasmids were linearized with BstI or BsbtI (NEB) and then transformed into Y1H Gold yeast strain cells and cultured on synthetic dextrose (SD) medium lacking Ura (SD/-Ura). The CDS of MdbHLH48 and MdWRKY6 were amplified and inserted into the pGADT7 vector, respectively. The recombinant pGADT7 plasmids were then transformed into Y1H (pAbAi-promoter) yeast strain cells, respectively. Transformed yeast cells were detected on SD lacking Leu (SD/-Leu) medium supplemented with a minimal inhibitory concentration of AbA for 3 to 4 d. The pGADT7 empty vector was used as a negative control. Details regarding the primers used for this assay are listed in Supplementary Table S2.
为了测试酵母中 MdbHLH48 与MdTFL1/MdFT1启动子的结合以及 MdWRKY6 与AFL1启动子的结合，使用 Matchmaker Gold Yeast One-Hybrid System Kit (Clontech) 进行 Y1H 测定。含有bHLH结合核心序列(GTAC)的MdTFL1和MdFT1启动子片段和含有W-box元件的AFL1启动子片段分别独立克隆到pAbAi载体中。将重组启动子-AbAi 质粒用 BstI 或 BsbtI (NEB) 线性化，然后转化至 Y1H Gold 酵母菌株细胞中，并在缺乏 Ura (SD/-Ura) 的合成葡萄糖 (SD) 培养基上培养。 MdbHLH48和MdWRKY6的CDS被扩增并分别插入到pGADT7载体中。然后将重组pGADT7质粒分别转化至Y1H（pAbAi启动子）酵母菌株细胞中。在补充有最小抑制浓度的 AbA 的 SD 缺乏 Leu (SD/-Leu) 培养基上检测转化的酵母细胞 3 至 4 d。 pGADT7空载体用作阴性对照。有关用于该测定的引物的详细信息列于补充表 S2中。

EMSA 欧洲航天局

The CDS of MdbHLH48 was cloned into the pMalC2 vector, which contains a maltose-binding protein (MBP) tag. The recombinant MBP protein and MdbHLH48-MBP fusion proteins were expressed in Escherichia coli BL21 (DE3) with 0.5 mM IPTG overnight at 18 °C and purified by affinity chromatography. For EMSA assays, oligonucleotide probes were synthesized and labeled with biotin by Sangon Biotechnology Co. Ltd. (Shanghai, China; see Supplementary Table S2). Unlabeled fragments of the same sequences served as competitive probes. The EMSA assay was performed using the LightShift Chemiluminescence EMSA kit (Beyotime, GS009) following the manufacturer's protocol.
MdbHLH48的 CDS 被克隆到 pMalC2 载体中，该载体包含麦芽糖结合蛋白 (MBP) 标签。重组MBP蛋白和MdbHLH48-MBP融合蛋白在大肠杆菌BL21（DE3）中用0.5 mM IPTG于18℃过夜表达，并通过亲和层析纯化。对于EMSA测定，寡核苷酸探针由生工生物技术有限公司（中国上海；见补充表S2 ）合成并用生物素标记。相同序列的未标记片段用作竞争性探针。 EMSA 测定是使用 LightShift 化学发光 EMSA 试剂盒（Beyotime，GS009）按照制造商的方案进行的。

Transient expression assays
瞬时表达测定

For the dual-luciferase transient expression assays, the promoter sequences (approximately 2.0 kb upstream of the start ATG) of MdTFL1, MdFT1, and AFL1 were separately amplified from ‘Fuji’ and recombined into the pGreenII 0800-LUC vector to generate reporters. The full-length CDS of MdbHLH48 and MdWRKY6 were separately amplified and recombined into the pGreenII 62-SK vector driven by the 35S promoter to generate the effectors. A. tumefaciens strain GV3101 (psoup) carrying the reporter and effector constructs was infiltrated into N. benthamiana leaves. Luciferase activity was detected using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, RG027) following the manufacturer's instructions. In each analysis, 5 independent N. benthamiana leaves were infiltrated and analyzed, and 3 biological replications were performed with quantification. The sequences of primers used in the transient expression assay are listed in Supplementary Table S2.
对于双荧光素酶瞬时表达测定， MdTFL1 、 MdFT1和AFL1的启动子序列（起始 ATG 上游约 2.0 kb）分别从“Fuji”扩增并重组到 pGreenII 0800-LUC 载体中以生成报告基因。 MdbHLH48和MdWRKY6的全长CDS被分别扩增并重组到由35S启动子驱动的pGreenII 62-SK载体中以产生效应子。携带报告子和效应子构建体的根癌农杆菌菌株 GV3101 (psoup) 被渗透到本塞姆氏烟草叶子中。按照制造商的说明，使用双荧光素酶报告基因检测试剂盒（Beyotime，RG027）检测荧光素酶活性。在每次分析中，5 个独立的N 。对Benthamiana叶子进行了渗透和分析，并进行了 3 次生物学重复并进行了定量。瞬时表达测定中使用的引物序列列于补充表S2中。

For the transient GUS expression assay, the MdTFL1 and MdFT1 promoter sequences (2 kb) were cloned into the pCAMBIA1381 vector, which contains a GUS tag sequence. The constructed pMdTFL1::GUS and pMdFT1::GUS were transferred to A. tumefaciens strain GV3101 cells and then vacuum infiltrated into apple leaves. The leaves harvested after 3 d of incubation were immersed in GUS staining buffer at 37 °C for 16 h and then immersed in 95% (v/v) ethanol to remove the chlorophyll. Histochemical staining for GUS was performed according to the method of Zhang et al. (2018). The GUS total protein was quantified using the Bradford protein assay kit method (Bio-Rad Company, United States).
对于瞬时 GUS 表达测定，将MdTFL1和MdFT1启动子序列 (2 kb) 克隆到 pCAMBIA1381 载体中，该载体包含 GUS 标签序列。将构建的pMdTFL1::GUS和pMdFT1 :: GUS转移到根癌农杆菌菌株GV3101细胞中，然后真空渗透到苹果叶中。将孵育3 d后收获的叶片浸入37℃的GUS染色缓冲液中16小时，然后浸入95％（v/v）乙醇中以去除叶绿素。 GUS的组织化学染色按照Zhang等的方法进行。 （2018） 。使用Bradford蛋白测定试剂盒方法(Bio-Rad公司，美国)对GUS总蛋白进行定量。

Subcellular localization assays
亚细胞定位测定

The 35S::MdWRKY6-GFP and 35S::GFP (positive control) constructs were introduced into A. tumefaciens strain GV3101 and then infiltrated into transgenic N. benthamiana leaves containing a nuclear localization signal (NLS-mCherry; Yang et al. 2020). Those infected tissues were analyzed 72 h after infiltration. Images were acquired using a confocal laser scanning microscope (Leica TCS SP8, Germany) with a 40×/1.4 water immersion objective. GFP excitation was performed using a 488-nm solid-state laser; fluorescence was detected at 498 to 540 nm; and the intensity and gain were 4.9% and 800, respectively. mCherry excitation was performed using a 552-nm solid-state laser; fluorescence was detected at 590 to 640 nm; and the intensity and gain were 9.1% and 800, respectively.
将35S::MdWRKY6-GFP和35S::GFP （阳性对照）构建体引入根癌农杆菌菌株 GV3101，然后渗透到含有核定位信号的转基因本塞姆氏烟草叶子中（NLS-mCherry； Yang 等人，2020 ） 。渗透后 72 小时对那些受感染的组织进行分析。使用具有 40×/1.4 水浸物镜的共焦激光扫描显微镜（Leica TCS SP8，德国）获取图像。使用 488 nm 固体激光器进行 GFP 激发；在 498 至 540 nm 处检测到荧光；强度和增益分别为4.9%和800。使用 552 nm 固态激光器进行 mCherry 激发；在 590 至 640 nm 处检测到荧光；强度和增益分别为9.1%和800。

Transcriptional activation activity assays
转录激活活性测定

The full-length CDS, as well as the various truncations of MdWRKY6, were separately fused with the GAL4 DNA-binding domain in the pGBKT7 vector. The fusion vectors were subsequently transformed into the yeast strain AH109. Transformation of the AH109 cells was performed according to the manufacturer's instructions. The empty pGBKT7 vector was used as the negative control. The transformed strains were grown and selected on SD medium lacking Trp (SD/-Trp) and SD medium lacking Trp, His, and Ade (SD/-Trp-His-Ade) with X-α-gal for 3 to 5 d at 30 °C.
全长 CDS 以及MdWRKY6的各种截短片段分别与 pGBKT7 载体中的 GAL4 DNA 结合结构域融合。随后将融合载体转化至酵母菌株AH109中。 AH109细胞的转化按照制造商的说明进行。空的pGBKT7载体用作阴性对照。将转化的菌株在缺乏Trp的SD培养基(SD/-Trp)和缺乏Trp、His和Ade的SD培养基(SD/-Trp-His-Ade)上与X-α-gal在3℃下生长和选择3至5天。 30°C。

Y2H assays Y2H 检测

Y2H assays were performed according to the manufacturer's Matchmaker Two-Hybrid System (Clontech). The coding regions of MdTFL1 and MdFT1 were cloned into the bait vector pGBKT7. Meanwhile, the coding regions of MdWRKY6 were recombined into the prey vector pGADT7. The amplification primers are listed in Supplementary Table S2. Bait and prey plasmids were cotransformed into Y2H Gold yeast strain and cultured on SD medium lacking Trp and Leu (SD/-Trp/-Leu) for 3 to 5 d at 30 °C. To test protein interactions, those colonies were selected on SD medium lacking Trp, Leu, His, and Ade (SD/-Trp/-Leu/-His/-Ade) with X-α-gal. The empty pGADT7 prey vector was used as a negative control.
Y2H 测定是根据制造商的 Matchmaker 双混合系统 (Clontech) 进行的。 MdTFL1和MdFT1的编码区被克隆到诱饵载体pGBKT7中。同时， MdWRKY6的编码区被重组到猎物载体pGADT7中。扩增引物列于补充表S2中。将诱饵和猎物质粒共转化到Y2H Gold酵母菌株中，并在缺乏Trp和Leu（SD/-Trp/-Leu）的SD培养基上于30℃培养3至5天。为了测试蛋白质相互作用，在缺乏 Trp、Leu、His 和 Ade (SD/-Trp/-Leu/-His/-Ade) 和 X-α-gal 的 SD 培养基上选择这些菌落。空的 pGADT7 猎物载体用作阴性对照。

BiFC

The MdTFL1, MdFT1, and MdWRKY6 coding regions were cloned into the YFP C-terminal (YFP^C) and YFP N-terminal (YFP^N) vectors, respectively. The primers used for vector construction are listed in Supplementary Table S2. The constructed vectors were transformed into A. tumefaciens strain GV3101 for transient infection in 5-wk-old N. benthamiana leaves. After coinfiltration for 48 h, fluorescence was observed by a confocal laser scanning microscope (Leica TCS SP8, Germany) with a water immersion objective (40×/1.4). YFP fluorescence was excited at 514 nm, and the emission from 522 to 560 nm was observed. Chlorophyll autofluorescence was detected between 670 and 720 nm.
将MdTFL1 、 MdFT1和MdWRKY6编码区分别克隆到YFP C端(YFP ^C )和YFP N端(YFP ^N )载体中。用于载体构建的引物列于补充表S2中。将构建的载体转化到根癌农杆菌菌株GV3101中，以瞬时感染5周龄的本塞姆氏烟草叶子。共滤48 h后，使用水浸物镜（40×/1.4）的共焦激光扫描显微镜（Leica TCS SP8，德国）观察荧光。 YFP 荧光在 514 nm 处激发，并观察到 ​​522 至 560 nm 的发射。在 670 至 720 nm 之间检测到叶绿素自发荧光。

Co-IP assay 共免疫沉淀检测

The CDS of MdTFL1 and MdFT1 were cloned into the pCAMBIA2300 vector with a GFP tag, respectively, and MdWRKY6 was cloned into the pCXSN vector with a Flag tag. The A. tumefaciens EHA105 strain carrying MdWRKY6-Flag was transiently coinfiltrated into N. benthamiana leaves with MdTFL1-GFP or MdFT1-GFP, respectively, to detect the in vivo interaction. The Co-IP assays were performed on N. benthamiana leaves as described (Li et al. 2016).
MdTFL1和MdFT1的CDS分别克隆到带有GFP标签的pCAMBIA2300载体中， MdWRKY6克隆到带有Flag标签的pCXSN载体中。将携带 MdWRKY6-Flag 的根癌农杆菌EHA105 菌株分别与 MdTFL1-GFP 或 MdFT1-GFP 瞬时共渗入本塞姆氏烟草叶子中，以检测体内相互作用。如所述（ Li et al. 2016 ），在本塞姆氏烟草叶子上进行 Co-IP 测定。

Firefly luciferase complementation imaging assay
萤火虫荧光素酶互补成像测定

The full-length CDSs of MdFT1, MdTFL1, and MdWRKY6 were separately inserted into the pCAMBIA1300-nLUC and pCAMBIA1300-cLUC vectors. Agrobacteria cells (strain GV3101) harboring the constructed vectors in different combinations were infiltrated into N. benthamiana leaves, and the infected plants were cultured at 22 °C for 3 d. Before LUC activity detection, the infiltrated leaves were sprayed with 1 mM of luciferin (D-Luciferin, Sodium Salt, Yeasen) and then kept in the darkness for 10 min to quench the background fluorescence. The LUC images were captured using a cooled CCD imaging apparatus (Lumazone Pylon 2048B, Princeton, United States). Each set of data consisted of 3 replicates, and 3 independent experiments were performed for each analysis.
将MdFT1 、 MdTFL1和MdWRKY6的全长CDS分别插入pCAMBIA1300-nLUC和pCAMBIA1300-cLUC载体中。将携带不同组合的构建载体的农杆菌细胞（菌株GV3101）浸润到本塞姆氏烟草叶子中，并将感染的植物在22℃培养3天。在检测 LUC 活性之前，将浸润的叶片喷洒 1 mM 荧光素（D-Luciferin、钠盐、Yeasen），然后在黑暗中放置 10 分钟以猝灭背景荧光。 LUC 图像是使用冷却 CCD 成像装置（Lumazone Pylon 2048B，普林斯顿，美国）捕获的。每组数据由 3 个重复组成，每个分析进行 3 个独立实验。

Statistical analyses 统计分析

Data were analyzed to calculate mean values and Se utilizing Microsoft Excel 2010 software (Microsoft Corp., United States). Statistical analyses were performed by Student's t test or by one-way ANOVA followed by Tukey’s test at a significance level of 0.05, using SPSS software (SPSS, Inc., Chicago, IL, United States). Figures were geminated by GraphPad Prism 8.0.2 software.
使用Microsoft Excel 2010软件（微软公司，美国）分析数据以计算平均值和Se。使用SPSS软件（SPSS，Inc.，芝加哥，伊利诺伊州，美国）通过Student's t检验或单向方差分析（ANOVA）进行统计分析，然后进行显着性水平为0.05的Tukey's检验。图形由 GraphPad Prism 8.0.2 软件生成。

Accession numbers 入藏号

Genes can be found with the following accession numbers: MdFT1 (MD12G1262000, AB161112), MdFT2 (MDP0000139278, AB458504), MdTFL1 (MD12G1023900, AB366639), MdTFL1a (MD14G1021100, AB366640), MdAFL1 (MD06G1129500), MdAP1 (MD13G1059200), MdSOC1 (MD02G1197400), MdbHLH48 (MD14G1064200), MdWRKY6 (MD05G1349800), and Histone H3 (LOC103406086).
可以通过以下登录号找到基因：MdFT1（MD12G1262000、AB161112）、MdFT2（MDP0000139278、AB458504）、MdTFL1（MD12G1023900、AB366639）、MdTFL1a（MD14G1021100、AB366640）、MdA FL1 (MD06G1129500)、MdAP1 (MD13G1059200)、MdSOC1 ( MD02G1197400)、MdbHLH48 (MD14G1064200)、MdWRKY6 (MD05G1349800) 和组蛋白 H3 (LOC103406086)。

Acknowledgments 致谢

We would like to thank Prof. X.F. Wang (Shandong Agricultural University, Taian, Shandong), Prof. S.L. Bai (Zhejiang University, Hangzhou, Zhejiang), and Dr. S. Fan (Northwest A&F University, Yangling, Shaanxi; National University of Singapore, Singapore) for their guidance on experiment operation. We also thank the Horticulture Science Research Center at College of Horticulture, NWAFU, for their technical support.
我们要感谢 XF Wang 教授（山东农业大学，山东泰安）、SL Bai 教授（浙江大学，浙江杭州）和 S. Fan 博士（西北农林科技大学，陕西杨凌；国立农业大学）新加坡，新加坡）对实验操作的指导。感谢西北农林科技大学园艺学院园艺科学研究中心的技术支持。

Author contributions 作者贡献

D.Z., L.X., and X.Z. conceived and designed the work. X.Z., S.W., and T.T. conducted the experiments. S.W., X.L., Y.L., and L.T. analyzed the data. K.S. did the English editing. X.Z., L.X., and D.Z. wrote the manuscript. N.A., J.M., and C.Z. discussed and modified the work content. All authors carefully revised and approved the manuscript.
DZ、LX 和 XZ 构思并设计了该作品。 XZ、SW 和 TT 进行了实验。 SW、XL、YL 和 LT 分析了数据。 KS 负责英文编辑。 XZ、LX 和 DZ 撰写了手稿。 NA、JM、CZ讨论并修改了工作内容。所有作者都仔细修改并批准了手稿。

Supplementary data 补充数据

The following materials are available in the online version of this article.
本文的在线版本中提供了以下材料。

Supplementary Figure S1. Relative expression of MdFT1, MdFT2, MdTFL1, and MdTFL1a in apple various tissues by RT-qPCR.
补充图S1 。 RT-qPCR 检测苹果不同组织中MdFT1 、 MdFT2 、 MdTFL1和MdTFL1a的相对表达量。

Supplementary Figure S2.MdFT1 and MdTFL1 are preferably expressed in the shoot apex, compared with MdFT2 and MdTFL1a, respectively.
补充图S2 。分别与MdFT2和MdTFL1a相比， MdFT1和MdTFL1优选在茎尖表达。

Supplementary Figure S3. Promoter analysis of MdTFL1 and MdFT1.
补充图S3 。 MdTFL1和MdFT1的启动子分析。

Supplementary Figure S4. Identification of the transgenic Arabidopsis, tomato, and apple calli overexpressing MdbHLH48.
补充图S4 。过表达MdbHLH48的转基因拟南芥、番茄和苹果愈伤组织的鉴定。

Supplementary Figure S5. Identification of the transgenic apple overexpressing MdbHLH48.
补充图S5 。过表达MdbHLH48的转基因苹果的鉴定。

Supplementary Figure S6. Phylogenetic analysis and alignment of the MdWRKY6 protein and the expression of MdWRKY6 in tissues and in apical buds during floral transition in apple.
补充图S6 。苹果花转变过程中 MdWRKY6 蛋白的系统发育分析和比对以及MdWRKY6在组织和顶芽中的表达。

Supplementary Figure S7. MdTFL1 interacts with MdWRKY6 protein via the C-terminal WRKY domain.
补充图S7 。 MdTFL1 通过 C 端 WRKY 结构域与 MdWRKY6 蛋白相互作用。

Supplementary Figure S8. Inhibition of MdTFL1-MdWRKY6 interaction by MdFT1 in firefly luciferase complementation imaging assay.
补充图S8 。在萤火虫荧光素酶互补成像测定中 MdFT1 对 MdTFL1-MdWRKY6 相互作用的抑制。

Supplementary Figure S9. Subcellular localization and transcriptional activity of MdWRKY6.
补充图S9 。 MdWRKY6 的亚细胞定位和转录活性。

Supplementary Figure S10. Verification of the transgenic Arabidopsis, tomato lines, and apple calli overexpressing MdWRKY6.
补充图S10 。验证过表达MdWRKY6的转基因拟南芥、番茄品系和苹果愈伤组织。

Supplementary Table S1. Read counts of each transcript model for RNA-seq data.
补充表S1 。 RNA-seq 数据的每个转录模型的读取计数。

Supplementary Table S2. Primer sequences used in this study.
补充表S2 。本研究中使用的引物序列。

Supplementary Materials and Methods.
补充材料和方法。

Funding 资金

This work was supported by the National Natural Science Foundation of China (32372657, 32072522, and 31872937), Shaanxi Apple Industry Science and Technology Project (2020zdzx03-01-04), the China Postdoctoral Science Foundation (2022M722618), the Rural Revitalization Project of Ningxia Hui Autonomous Region (2021CFSF0013), and the China Agriculture Research System of MOF and MARA (CARS-27).
该工作得到国家自然科学基金项目（32372657、32072522、31872937）、陕西省苹果产业科技项目（2020zdzx03-01-04）、中国博士后科学基金项目（2022M722618）、农村振兴项目的资助宁夏回族自治区（2021CFSF0013），以及财政部和农业农村部中国农业研究系统（CARS-27）。

Dive Curated Terms 潜水策划术语

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
以下表型、基因型和功能术语对本文所述的工作具有重要意义：

• CRY2 Gramene: AT1G04400 CRY2 格拉烯：AT1G04400

• CRY2 Araport: AT1G04400 CRY2 阿拉波特：AT1G04400

• PIF4 Gramene: AT2G43010 PIF4 格拉烯：AT2G43010

• PIF4 Araport: AT2G43010 PIF4 阿拉波特：AT2G43010

• WRKY34 Gramene: AT4G26440
  WRKY34 格拉烯：AT4G26440

• WRKY34 Araport: AT4G26440
  WRKY34 阿拉波特：AT4G26440

• WRKY71 Gramene: AT1G29860
  WRKY71 格拉烯：AT1G29860

• WRKY71 Araport: AT1G29860
  WRKY71 阿拉波特：AT1G29860

• FT Gramene: AT1G65480 FT Gramene：AT1G65480

• FT Araport: AT1G65480 英国《金融时报》阿拉波特：AT1G65480

• WRKY13 Gramene: AT4G39410
  WRKY13 格拉烯：AT4G39410

• WRKY13 Araport: AT4G39410
  WRKY13 阿拉波特：AT4G39410

• FLC Gramene: AT5G10140 FLC 格拉烯：AT5G10140

• FLC Araport: AT5G10140 FLC 阿拉波特：AT5G10140

• WRKY75 Gramene: AT5G13080
  WRKY75 格拉烯：AT5G13080

• WRKY75 Araport: AT5G13080
  WRKY75 阿拉波特：AT5G13080

• AFL1 Gramene: AT3G28270 AFL1 格拉烯：AT3G28270

• AFL1 Araport: AT3G28270 AFL1 阿拉波特：AT3G28270

References 参考