College of Horticulture, Qingdao Agricultural University, Qingdao, 266109, China. carotenoid-derived apple fruit coloration 青岛农业大学园艺学院，中国青岛，266109。 类胡萝卜素衍生苹果果实着色
Dongjie , Yuchen , Kun Jia , Benchang Huang , Qingyuan Dang , Huimin Wang , Xinyuan Wang , Chunyu Li , Yugang Zhang , Jiyun Nie , Yongbing Yuan Dongjie , Yuchen , Kun Jia , Benchang Huang , Qingyuan Dang , Huimin Wang , Xinyuan Wang , Chunyu Li , Yugang Zhang , Jiyun Nie , Yongbing Yuan
Laboratory of Quality & Safety Risk Assessment for Fruit (Qingdao), Ministry of Agriculture and Rural Affairs/National Technology Centre for Whole Process Quality Control of FSEN Horticultural Products (Qingdao)/Qingdao Key Lab of Modern Agriculture Quality and Safety Engineering, Qingdao, 266109 , China. 农业农村部果品质量安全风险评估实验室（青岛）/国家FSEN园艺产品全程质量控制技术中心（青岛）/青岛市现代农业质量安全工程重点实验室，中国青岛，266109。
6 The author responsible for distribution of materials integral to the findings presented in this 7 article in accordance with the policy described in the Instructions for Authors 8 (https://academic.oup.com/plphys/pages/General-Instructions) is Yongbing Yuan. 6 根据《作者须知》( https://academic.oup.com/plphys/pages/General-Instructions) 中所述的政策，负责分发与本文 7 中的研究结果有关的材料的作者是袁勇兵。
Abscisic acid activates transcription factor module MdABI5-MdMYBS1 during 脱落酸可激活转录因子模块 MdABI5-MdMYBS1。
Short Title: ABI5-MYBS1 regulates carotenoid and ABA synthesis 简短标题：ABI5-MYBS1 调控类胡萝卜素和 ABA 的合成
Abstract 摘要
Carotenoids are major pigments contributing to fruit coloration. We previously reported that the apple (Malus domestica Borkh.) mutant fruits of 'Beni Shogun' and 'Yanfu 3' show a marked difference in fruit coloration. However, the regulatory mechanism underlying this phenomenon remains unclear. In this study, we determined that carotenoid is the main factor influencing fruit flesh color. We identified an R1-type MYB transcription factor, MdMYBS1, which was found to be highly associated with carotenoids and abscisic acid (ABA) contents of apple fruits. Overexpression of MdMYBS1 promoted, and silencing of MdMYBS1 repressed, -branch carotenoids synthesis and ABA accumulation. MdMYBS1 regulates carotenoid biosynthesis by directly activating the major carotenoid biosynthetic genes encoding phytoene synthase (MdPSY2-1) and lycopene -cyclase (MdLCYb). 9-cis-epoxycarotenoid dioxygenase 1 (MdNCED1) contributes to ABA biosynthesis, and MdMYBS1 enhances endogenous ABA accumulation by activating the MdNCED1 promoter. In addition, the basic leucine zipper domain transcription 类胡萝卜素是导致果实着色的主要色素。我们以前曾报道过苹果（Malus domestica Borkh.）突变体果实'贝尼将军'和'盐富 3 号'在果实着色方面表现出明显的差异。然而，这一现象背后的调控机制仍不清楚。在本研究中，我们确定类胡萝卜素是影响果肉颜色的主要因素。我们发现一个 R1 型 MYB 转录因子 MdMYBS1 与苹果果实的类胡萝卜素和脱落酸（ABA）含量高度相关。过表达 MdMYBS1 促进 - 分支类胡萝卜素的合成和 ABA 的积累，而沉默 MdMYBS1 则抑制类胡萝卜素的合成和 ABA 的积累。MdMYBS1通过直接激活编码植物烯合成酶（MdPSY2-1）和番茄红素 -环化酶（MdLCYb）的主要类胡萝卜素生物合成基因来调节类胡萝卜素的生物合成。9-cis-epoxycarotenoid dioxygenase 1（MdNCED1）有助于 ABA 的生物合成，MdMYBS1 通过激活 MdNCED1 启动子来增强内源 ABA 的积累。此外，碱性亮氨酸拉链结构域转录
factor ABSCISIC ACID-INSENSITIVE5 (MdABI5) was identified as an upstream activator of MdMYBS1, which promotes carotenoid and ABA accumulation. Furthermore, ABA promotes carotenoid biosynthesis and enhances MdMYBS1 and MdABI5 promoter activities. Our findings demonstrate that the MdABI5-MdMYBS1 cascade activated by ABA regulates carotenoid-derived fruit coloration and ABA accumulation in apple, providing avenues in breeding and planting for improvement of fruit coloration and quality. 因子 ABSCISIC ACID-INSENSITIVE5（MdABI5）被确定为 MdMYBS1 的上游激活因子，可促进类胡萝卜素和 ABA 的积累。此外，ABA 促进类胡萝卜素的生物合成，并增强 MdMYBS1 和 MdABI5 的启动子活性。我们的研究结果表明，ABA 激活的 MdABI5-MdMYBS1 级联调节类胡萝卜素衍生的苹果果实着色和 ABA 积累，为育种和种植提供了改善果实着色和品质的途径。
Carotenoids are major pigments contributing to the yellow, orange, red, and other coloration of horticultural fruits and vegetables (Yuan et al., 2015). In plants, carotenoids play vital roles in plant growth and development, and biotic interactions such as photoprotections (Domonkos et al., 2013; Sandmann, 2021), precursors of abscisic acid (ABA), strigolactones and bioactive apocarotenoids (Ramel et al., 2012; Moreno et al., 2021). Furthermore, carotenoids are antioxidants that benefit human health (Fiedor and Burda, 2014). Therefore, as our emphasis on quality of life has increased, so has the need for cultivating new plant varieties rich in carotenoids via plant genetic engineering. 类胡萝卜素是园艺水果和蔬菜呈现黄色、橙色、红色和其他颜色的主要色素（Yuan 等人，2015 年）。在植物中，类胡萝卜素在植物生长发育和生物相互作用中发挥着重要作用，例如光保护作用（Domonkos 等人，2013 年；Sandmann，2021 年）、脱落酸（ABA）的前体、绞股蓝内酯和生物活性类胡萝卜素（Ramel 等人，2012 年；Moreno 等人，2021 年）。此外，类胡萝卜素还是有益于人类健康的抗氧化剂（Fiedor 和 Burda，2014 年）。因此，随着我们对生活质量的重视程度越来越高，通过植物基因工程培育富含类胡萝卜素的植物新品种的需求也越来越大。
Carotenoids are isoprenoid compounds synthesized by various metabolic enzymes (Sun et al., 类胡萝卜素是由各种代谢酶合成的异戊二烯化合物（Sun 等人）、
2018). The diversity of carotenoid pigments is influenced by carotenoid biosynthetic genes. Multiple phytoene synthases (PSYs) influence carotenoid content in several species such as tomato (Solanum lycopersicum) (Fraser et al., 2002), maize (Zea mays) (Li et al., 2008), Arabidopsis thaliana (Rodriguez-Villalon et al., 2009), rice (Oryza sativa) (Bai et al., 2016), and pepper (Capsicum spp.) (Jeong et al., 2019; Jang et al., 2020). Overexpressing MdPSY2-1 and MdPSY1 increases carotenoid contents in apple (Malus domestica Borkh.) (Dang et al., 2021; Ampomah-Dwamena et al., 2022). Moreover, downregulating lycopene B-cyclase (ClLCYB) contributes to lycopene accumulation and red flesh coloration in watermelon (Citrullus lanatus) (Zhang et al., 2020). 2018).类胡萝卜素色素的多样性受类胡萝卜素生物合成基因的影响。在番茄（Solanum lycopersicum）（Fraser 等人，2002 年）、玉米（Zea mays）（Li 等人，2008 年）、拟南芥（Rodriguez-Villalon 等人，2009 年）等多个物种中，多种植物烯合成酶（PSYs）影响类胡萝卜素的含量、2008）、拟南芥（Rodriguez-Villalon 等人，2009）、水稻（Oryza sativa）（Bai 等人，2016）和辣椒（Capsicum spp.）（Jeong 等人，2019；Jang 等人，2020）。过表达 MdPSY2-1 和 MdPSY1 会增加苹果（Malus domestica Borkh.）中类胡萝卜素的含量（Dang 等人，2021 年；Ampomah-Dwamena 等人，2022 年）。此外，下调番茄红素 B-环化酶（ClLCYB）有助于番茄红素的积累和西瓜（Citrullus lanatus）的红肉着色（Zhang 等人，2020 年）。
The MYB transcription factor (TF) superfamily is grouped into four subfamilies including R1/R2-MYB, R2R3-MYB, R1R2R3-MYB and R1R2R3R1/R2-MYB depending on the number of MYB repeats. MYB TFs play vital roles in growth, development, phytohormone synthesis, and stress in diverse plant species (Dubos et al., 2010; Wu et al., 2022). Several MYB TFs modulate carotenoid biosynthesis. The Reduced Carotenoid Pigmentation 1 (RCP1) as an R2R3-MYB positively modulates carotenoid biosynthesis in Mimulus lewisii flowers (Sagawa et al., 2016). WHITE PETAL1 (WP1) as an R2R3-MYB enhances carotenoid production by promoting lycopene -cyclase (MtLYCe) and lycopene B-cyclase (MtLYCb) expression in Medicago truncatula flowers (Meng et al., 2019). SIMYB72 and SIMYB117 regulate carotenoid biosynthesis in tomato fruits by modulating carotenogenic genes (Wu et al., 2020; Tyagi et al., 2022). CrMYB68 inhibits - and -branch carotenoid biosynthesis via repressing b-carotene hydroxylases and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) expression in citrus (Zhu et al., 2017). By contrast, 4 MYB 转录因子（TF）超家族根据 MYB 重复的数量分为四个亚家族，包括 R1/R2-MYB、R2R3-MYB、R1R2R3-MYB 和 R1R2R3R1/R2-MYB。MYB TFs 在不同植物物种的生长、发育、植物激素合成和胁迫中发挥着重要作用（Dubos 等人，2010 年；Wu 等人，2022 年）。一些 MYB TFs 可调节类胡萝卜素的生物合成。类胡萝卜素色素沉着减少 1（RCP1）作为一种 R2R3-MYB，能积极调节 Mimulus lewisii 花中类胡萝卜素的生物合成（Sagawa 等，2016 年）。白色 PETAL1（WP1）作为一种 R2R3-MYB，通过促进番茄红素 -环化酶（MtLYCe）和番茄红素 B-环化酶（MtLYCb）在 Medicago truncatula 花中的表达，提高类胡萝卜素的产量（Meng 等人，2019 年）。SIMYB72 和 SIMYB117 通过调节类胡萝卜素生成基因来调控番茄果实中类胡萝卜素的生物合成（Wu 等人，2020；Tyagi 等人，2022）。CrMYB68 通过抑制柑橘中 b-胡萝卜素羟化酶 和 9-顺式环氧类胡萝卜素二氧酶 5（CrNCED5）的表达，抑制 - 和 - 分支类胡萝卜素的生物合成（Zhu 等，2017 年）。相比之下，4
AdMYB7 promotes carotenoid production by directly enhancing expression in kiwifruit (Actinidia deliciosa) (Ampomah-Dwamena et al., 2019). Therefore, MYB subfamilies have multiple roles in regulating carotenoid biosynthesis in diverse plant species. AdMYB7 通过直接提高 在猕猴桃（Actinidia deliciosa）中的表达，促进类胡萝卜素的产生（Ampomah-Dwamena 等人，2019 年）。因此，MYB 亚家族在调节不同植物物种中类胡萝卜素的生物合成方面具有多重作用。
Abscisic acid (ABA), which is produced by 9-cis-epoxycarotenoid dioxygenase (NCED) cyclizing of violaxanthin or neoxanthin, is an important phytohormone that plays key roles in plant growth and development (Chen et al., 2020; Kavi Kishor et al., 2022). The enzyme NCED is the vital rate-limiting step for ABA biosynthesis (Frey et al., 2012; Lang et al., 2021). Silencing SINCED1 reduces ABA levels and increases levels of lycopene and -carotene in tomato fruits (Sun et al., 2012a, b). Overexpressing PpNCED1 and PpNCED5 enhances ABA levels in peach (Prunus persica L. Batsch) calli (Wang et al., 2021). Several TFs participate in ABA-mediated carotenoid biosynthesis. ABA-DEFICIENT4 (ABA4) controls the accumulation of 9-cis-violaxanthin in Arabidopsis (Perreau et al., 2020). However, little is known about ABA-regulated carotenoid biosynthesis in apple fruit. 脱落酸（ABA）由 9-顺式环氧类胡萝卜素二氧酶（NCED）环化 violaxanthin 或 neoxanthin 生成，是一种重要的植物激素，在植物生长和发育过程中发挥关键作用（Chen 等人，2020 年；Kavi Kishor 等人，2022 年）。NCED 酶是 ABA 生物合成的重要限速步骤（Frey 等人，2012；Lang 等人，2021）。沉默 SINCED1 可降低番茄果实中的 ABA 水平，提高番茄红素和 - 胡萝卜素的水平（Sun 等，2012a, b）。过表达 PpNCED1 和 PpNCED5 可提高桃（Prunus persica L. Batsch）胼胝体中的 ABA 水平（Wang 等人，2021 年）。一些 TFs 参与了 ABA 介导的类胡萝卜素生物合成。ABA-DEFICIENT4（ABA4）控制拟南芥中 9-顺式紫杉素的积累（Perreau 等人，2020 年）。然而，人们对苹果果实中受 ABA 调节的类胡萝卜素生物合成知之甚少。
Fruit flesh color is mainly determined by amounts of carotenoids (Ampomah-Dwamena et al., 2012; Dang et al., 2021). Little is known about the regulatory network of carotenoid biosynthesis and accumulation in apple fruit. We previously reported that there was a marked difference in total carotenoid content of apple mature fruits between 'Beni Shogun' and 'Yanfu 3' (Dang et al., 2021). Here, we identify an R1-type MdMYBS1 that promotes carotenoid accumulation and fruit coloration by directly activating MdPSY2-1 and MdLCYb expression. Furthermore, MdMYBS1 activates MdNCED1 expression to enhance ABA accumulation, and ABA induces MdMYBS1 transcription via a basic leucine zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (MdABI5). 5 果肉颜色主要由类胡萝卜素的含量决定（Ampomah-Dwamena 等人，2012 年；Dang 等人，2021 年）。人们对苹果果实中类胡萝卜素生物合成和积累的调控网络知之甚少。我们以前曾报道过，'贝尼将军'和'盐富 3 号'苹果成熟果实中类胡萝卜素的总含量存在明显差异（Dang 等人，2021 年）。在这里，我们发现了一种 R1 型 MdMYBS1，它通过直接激活 MdPSY2-1 和 MdLCYb 的表达来促进类胡萝卜素的积累和果实着色。此外，MdMYBS1 还能激活 MdNCED1 的表达以增强 ABA 的积累，ABA 通过碱性亮氨酸拉链转录因子 ABSCISIC ACID-INSENSITIVE5 (MdABI5)诱导 MdMYBS1 的转录。5
Our findings thus provide insights into the regulatory mechanism of the 'MdABI5-MdMYBS1' module underlying carotenoid biosynthesis and new opportunities for genetic improvement of fruit quality and coloration. 因此，我们的研究结果有助于深入了解类胡萝卜素生物合成所依赖的 "MdABI5-MdMYBS1 "模块的调控机制，并为果实品质和着色的遗传改良提供了新的机遇。
Results 成果
Carotenoid content in fruits showing different coloration 不同颜色水果中的类胡萝卜素含量
We previously reported that total carotenoid content of ripening fruit flesh was markedly higher in 'Beni Shogun' than in 'Yanfu 3' (Dang et al., 2021). The flesh of ripening 'Beni Shogun' fruit was orange, whereas that of 'Yanfu 3' was yellow (Figure 1A). Carotenoids and flavonoids play vital roles in fruit flesh color (Dang et al., 2021; Han et al., 2022). The total carotenoid content in fruit flesh significantly differed between 'Beni Shogun' and 'Yanfu 3' during fruit development at 30 and 0 days before fruit ripening (DBFR) but total flavonoid content did not (Figure 1A and Supplemental Figure S1), indicating that carotenoid is the main factor influencing flesh color of apple fruit. 我们以前曾报道过，成熟果肉中类胡萝卜素的总含量'贝尼将军'明显高于'盐富 3 号'（Dang 等人，2021 年）。成熟的'贝尼将军'果肉呈橙色，而'盐富 3 号'果肉呈黄色（图 1A）。类胡萝卜素和类黄酮对果肉颜色起着重要作用（Dang 等，2021 年；Han 等，2022 年）。在果实成熟前 30 天和 0 天（DBFR）的果实发育过程中，'贝尼将军'和'盐富 3 号'果肉中类胡萝卜素的总含量存在显著差异，但类黄酮的总含量没有显著差异（图 1A 和补充图 S1），表明类胡萝卜素是影响苹果果肉颜色的主要因素。
We detected six carotenoid compounds including phytoene, -carotene, -cryptoxanthin, violaxanthin, neoxanthin and lutein in apple fruit flesh using LC-MS/MS (Supplemental Figure S2A). However, zeaxanthin, antheraxanthin, -carotene, and -cryptoxanthin were not detected (Supplemental Figure S3). The major carotenoids in fruit flesh of 'Beni Shogun' were phytoene and -carotene, and of 'Yanfu 3 ' were -carotene and -cryptoxanthin (Figure 1, B-G). Phytoene level was high in 'Beni Shogun' fruit flesh at 30 and 0 DBFR, but this compound was not detected in 'Yanfu 3' fruit flesh at 60 or 30 DBFR or in 'Beni Shogun' fruit flesh at 60 DBFR (Figure 1B). 通过 LC-MS/MS，我们在苹果果肉中检测到了六种类胡萝卜素化合物，包括植物黄素、 -胡萝卜素、 -隐黄素、小叶黄素、新黄素和叶黄素（补充图 S2A）。但是，没有检测到玉米黄质、花生黄质、 - 胡萝卜素和 - 隐黄质（补充图 S3）。贝尼将军 "果肉中的主要类胡萝卜素是植物色素和 -胡萝卜素，"盐富 3 号 "果肉中的主要类胡萝卜素是 -胡萝卜素和 -隐黄素（图 1，B-G）。贝尼将军 "果肉在 30 DBFR 和 0 DBFR 时的植物烯含量较高，但 "盐富 3 号 "果肉在 60 DBFR 和 30 DBFR 时以及 "贝尼将军 "果肉在 60 DBFR 时均未检测到该化合物（图 1B）。
Levels of -carotene (Figure 1C) and -cryptoxanthin (Figure 1D) were markedly higher in 'Beni Shogun' than in 'Yanfu 3' at 60, 30 and 0 DBFR, with differences of two- to four-fold. Neoxanthin (Figure 1F) and lutein (Figure 1G) contents were much lower in 'Beni Shogun' than in 'Yanfu 3' during fruit ripening, whereas violaxanthin level was lower at 60 DBFR and higher at 0 DBFR in 'Beni Shogun' than in 'Yanfu 3' (Figure 1E). -胡萝卜素（图 1C）和 -隐黄素（图 1D）的含量在 60、30 和 0 DBFR 时，'贝尼将军'明显高于'盐富 3 号'，相差 2 至 4 倍。在果实成熟过程中，'贝尼将军'的新黄质（图 1F）和叶黄素（图 1G）含量远低于'盐富 3 号'，而'贝尼将军'在 60 DBFR 和 0 DBFR 时的中黄质含量低于'盐富 3 号'（图 1E）。
In addition, because is derived from the cleavage of carotenoid precursors, we examined endogenous ABA content using LC-MS/MS (Supplemental Figure S2B). ABA level was significantly (three- to six -fold) higher in 'Beni Shogun' than in 'Yanfu 3' fruit flesh at 60,30 and 0 DBFR (Figure 1H). 此外，由于 来自类胡萝卜素前体的裂解，我们使用 LC-MS/MS 检测了内源 ABA 的含量（补充图 S2B）。在 60、30 和 0 DBFR 条件下，'贝尼将军'果肉中的 ABA 含量明显高于'盐富 3 号'果肉（3 至 6 倍）（图 1H）。
MdMYBS1 is a candidate regulator of carotenoid and ABA accumulation MdMYBS1 是类胡萝卜素和 ABA 积累的候选调控因子
To identify the key regulators of carotenoid biosynthesis and ABA accumulation in apple fruit, 18 RNA sequencing (RNA-seq) libraries were sequenced from the flesh of 'Beni Shogun' and 'Yanfu 3' fruits at 60, 30, and 0 DBFR (Supplemental Figure S4, A-C). Differentially expressed genes (DEGs) were defined with false discovery rate (FDR) , and expression variation ratio . There were 2569 total DEGs, 446 up-regulated DEGs and 441 down-regulated DEGs for the three time points of fruit development (Supplemental Figure S4, D-F). We identified one up-regulated MYB-related TF gene (MdMYBS1, MDO0G1169600) from 446 up-regulated DEGs (Supplemental Figure S4E) and two down-regulated MYB-related TF genes (PHL11, MD00G1000500; MYB88, MD16G1076100) from 441 down-regulated DEGs (Supplemental Figure S4F). Moreover, because there was a significant difference in carotenoids and ABA levels between 'Beni Shogun' and 为了确定苹果果实中类胡萝卜素生物合成和 ABA 积累的关键调控因子，对'贝尼将军'和'盐富 3 号'果实在 60、30 和 0 DBFR 时的果肉进行了 18 个 RNA 测序（RNA-seq）文库测序（补充图 S4，A-C）。差异表达基因（DEGs）用错误发现率（FDR） ，表达变异比 。在果实发育的三个时间点中，共有 2569 个 DEGs，446 个上调 DEGs 和 441 个下调 DEGs（补充图 S4，D-F）。我们从 446 个上调 DEGs 中发现了一个上调的 MYB 相关 TF 基因（MdMYBS1，MDO0G1169600）（补图 S4E），从 441 个下调 DEGs 中发现了两个下调的 MYB 相关 TF 基因（PHL11，MD00G1000500；MYB88，MD16G1076100）（补图 S4F）。此外，由于类胡萝卜素和 ABA 水平在 "贝尼将军 "和
'Yanfu 3' fruits at 30 and 0 DBFR, we identified three up-regulated MYB-related TF genes from 2736 up-regulated DEGs (Supplemental Figure S4E) and 10 down-regulated MYB-related TF genes from 3886 down-regulated DEGs (Supplemental Figure S4F) in the 30 DBFR vs. O DBFR comparison (Supplemental Figure S5). 在 30 DBFR 和 0 DBFR 条件下，我们从 2736 个上调 DEGs 中发现了 3 个上调的 MYB 相关 TF 基因（补充图 S4E），从 3886 个下调 DEGs 中发现了 10 个下调的 MYB 相关 TF 基因（补充图 S4F）（补充图 S5）。
Further RT-qPCR assays showed that MdMYBS1 transcript level was more highly correlated with total carotenoid, phytoene, -carotene, -cryptoxanthin and ABA contents compared to the transcript levels of other MYB TF genes (Figure 1i and Supplemental Table S1). MdMYBS1 was also expressed in the leaf and flower (Supplemental Figure S6). Furthermore, expression of MdMYBS1, which is high in orange/yellow fruit flesh (i.e., 'Gala') and low in white fruit flesh (i.e., 'Granny Smith'), was related with carotenoid abundance and fruit coloration in 13 Malus accessions (Supplemental Figure S7). We therefore chose MdMYBS1 for further analysis. 进一步的 RT-qPCR 分析表明，与其他 MYB TF 基因的转录水平相比，MdMYBS1 的转录水平与类胡萝卜素总量、植物色素、 -胡萝卜素、 -隐黄素和 ABA 的含量有更高的相关性（图 1i 和补充表 S1）。MdMYBS1 也在叶片和花中表达（补充图 S6）。此外，MdMYBS1 在橙色/黄色果肉（即'Gala'）中的表达量较高，而在白色果肉（即'Granny Smith'）中的表达量较低，这与 13 个 Malus 品种中类胡萝卜素的丰度和果实着色有关（补充图 S7）。因此，我们选择 MdMYBS1 作进一步分析。
MdMYBS1 positively regulates carotenoid and ABA accumulation MdMYBS1 积极调控类胡萝卜素和 ABA 的积累
MdMYBS1, an R1-type MYB TF, comprises 328 amino acids with a central SANT/MYB DNA-binding domain and an ZnF_C2HC domain, as predicted by the SMART website (Figure 2A). Phylogenetic analysis indicated the MdMYBS1 amino acid sequence was most similar to that of the Arabidopsis MYB TF KUODA1 (KUA1) (71.56% similarity) (Figure 2B) (Lu et al., 2014; Huang et al., 2015). The DNA-binding region of MdMYBS1 contains an SHAQKYF motif, which is conserved in R1-type MYB TFs such as AtKUA1, OsMYBS3, and OsMYBS2. AtKUA1 and OsMYBS3 function as transcriptional repressors and contain an EAR motif (LxLxL), whereas OsMYBS2 is a transcriptional activator and lacks an EAR motif (Lu et al., 2002); MdMYBS1 also lacks an EAR motif (Figure 2C). Subcellular 根据 SMART 网站的预测，MdMYBS1 是一种 R1 型 MYB TF，由 328 个氨基酸组成，具有一个中央 SANT/MYB DNA 结合结构域和一个 ZnF_C2HC 结构域（图 2A）。系统发育分析表明，MdMYBS1 的氨基酸序列与拟南芥 MYB TF KUODA1（KUA1）的氨基酸序列最为相似（相似度为 71.56%）（图 2B）（Lu 等，2014；Huang 等，2015）。MdMYBS1 的 DNA 结合区包含一个 SHAQKYF 基序，该基序在 AtKUA1、OsMYBS3 和 OsMYBS2 等 R1 型 MYB TF 中是保守的。AtKUA1 和 OsMYBS3 是转录抑制因子，含有一个 EAR 基序（LxLxL），而 OsMYBS2 是转录激活因子，缺乏 EAR 基序（Lu 等人，2002 年）；MdMYBS1 也缺乏 EAR 基序（图 2C）。亚细胞
localization analysis using MdMYBS1-GFP fusion protein showed that MdMYBS1 is localized in the nucleus (Figure 2D). 利用 MdMYBS1-GFP 融合蛋白进行的定位分析表明，MdMYBS1 定位于细胞核中（图 2D）。
We performed a transient expression assay to overexpress or silence MdMYBS1 in 'Granny Smith' fruit to test whether MdMYBS1 is involved in carotenoid and ABA accumulation. We constructed 35S:MdMYBS1 (MdMYBS1-OVX) vectors for gene overexpression and TRV:MdMYBS1 (MdMYBS1-TRV) vectors for gene silencing; the empty vector pRI101-flag (P101F) served as an overexpression control, and TRV1/TRV2 (TRV) served as a silencing control (Figure 3A). Deep yellow coloration, an indicator of carotenoid content, was observed in MdMYBS1-OVX fruits (Figure 3B). MdMYBS1 was highly overexpressed in MdMYBS1-OVX fruits and highly silenced in MdMYBS1-TRV fruits (Figure 3C). Significantly higher levels of total carotenoid, phytoene, -carotene, -cryptoxanthin, violaxanthin and ABA were detected in MdMYBS1-OVX yet much lower in MdMYBS1-TRV fruits (Figure 3, D-F) than those of the controls, whereas the neoxanthin level followed an opposite trend (Figure 3E). 我们进行了瞬时表达试验，在'Granny Smith'果实中过表达或沉默 MdMYBS1，以检验 MdMYBS1 是否参与类胡萝卜素和 ABA 的积累。我们构建了35S:MdMYBS1（MdMYBS1-OVX）载体用于基因过表达，TRV:MdMYBS1（MdMYBS1-TRV）载体用于基因沉默；空载体pRI101-flag（P101F）作为过表达对照，TRV1/TRV2（TRV）作为沉默对照（图3A）。在 MdMYBS1-OVX 果实上观察到类胡萝卜素含量指标--深黄色（图 3B）。在 MdMYBS1-OVX 果实中，MdMYBS1 被高度过表达，而在 MdMYBS1-TRV 果实中，MdMYBS1 被高度沉默（图 3C）。在 MdMYBS1-OVX 果实中检测到的总类胡萝卜素、植物烯、 -胡萝卜素、 -隐黄素、violaxanthin 和 ABA 含量显著高于对照组，而在 MdMYBS1-TRV 果实中则远低于对照组（图 3，D-F），而新黄质含量则呈相反趋势（图 3E）。
We also overexpressed or silenced MdMYBS1 in apple calli and plants. We constructed the RNAi vector pRI101-AN-MdMYBS1 (MdMYBS1-RNAi) and used it for stable transformation; the empty vector pRI101-AN (P101R) served as a control (Figure 3A). MdMYBS1-OVX apple calli (Figure 3G) and apple plants (Figure 3L) showed a deeper yellow color than controls. MdMYBS1 was highly overexpressed in MdMYBS1-OVX calli (Figure 3H) and leaves (Figure 3M) and highly silenced in MdMYBS1-RNAi calli (Figure 3H). Levels of total carotenoid, phytoene, -carotene, -cryptoxanthin, violaxanthin and ABA were markedly higher in MdMYBS1-OVX calli (Figure 3, I-K) and apple leaves (Figure 3, N-P), and much lower in MdMYBS1-RNAi calli than in the controls 9 我们还在苹果胼胝体和植株中过表达或沉默了 MdMYBS1。我们构建了 RNAi 载体 pRI101-AN-MdMYBS1（MdMYBS1-RNAi）并将其用于稳定转化；空载体 pRI101-AN (P101R) 作为对照（图 3A）。与对照组相比，MdMYBS1-OVX 苹果胼胝体（图 3G）和苹果植株（图 3L）显示出更深的黄色。MdMYBS1 在 MdMYBS1-OVX 胼胝体（图 3H）和叶片（图 3M）中高度过表达，而在 MdMYBS1-RNAi 胼胝体（图 3H）中则高度沉默。在 MdMYBS1-OVX 胼胝体（图 3，I-K）和苹果叶片（图 3，N-P）中，总类胡萝卜素、植物烯、 -胡萝卜素、 -隐黄素、violaxanthin 和 ABA 的水平明显高于对照组，而在 MdMYBS1-RNAi 胼胝体中则远低于对照组 9。
(Figure 3, I-K). However, the neoxanthin level was markedly lower in MdMYBS1-OVX calli (Figure 3J) and apple leaves (Figure 30), and higher in MdMYBS1-RNAi calli than in the controls (Figure 3J). (图 3，I-K）。然而，与对照组相比，MdMYBS1-OVX 胼胝体（图 3J）和苹果叶片（图 30）中的新黄质含量明显较低，而 MdMYBS1-RNAi 胼胝体中的新黄质含量较高（图 3J）。
Furthermore, we transformed MdMYBS1 into Micro-Tom tomato for heterologous expression. MdMYBS1-OVX tomato flower and fruits showed a deeper yellow color than controls (Figure 3Q). MdMYBS1 was highly expressed in MdMYBS1-OVX tomato fruits (Figure 3R). Levels of total carotenoid, phytoene, lycopene, -carotene and ABA were markedly higher in MdMYBS1-OVX tomato fruits (Figure 3, S-U). Together, these results demonstrate that MdMYBS1 activates accumulation of -branch carotenoids and endogenous ABA. 此外，我们还将 MdMYBS1 转化到 Micro-Tom 番茄中进行异源表达。与对照组相比，MdMYBS1-OVX 番茄的花和果实呈现出更深的黄色（图 3Q）。MdMYBS1 在 MdMYBS1-OVX 番茄果实中高表达（图 3R）。在 MdMYBS1-OVX 番茄果实中，总类胡萝卜素、植物色素、番茄红素、 - 胡萝卜素和 ABA 的水平明显较高（图 3，S-U）。这些结果表明，MdMYBS1 激活了 - 分支类胡萝卜素和内源 ABA 的积累。
MdMYBS1 regulates carotenoid biosynthesis by activating MdPSY2-1 and MdLCYb MdMYBS1 通过激活 MdPSY2-1 和 MdLCYb 来调节类胡萝卜素的生物合成
Next, we reasoned that MdMYBS1 might regulate carotenoid accumulation by modulating carotenoid biosynthetic genes. We identified carotenoid biosynthesis genes in RNA-seq data (Supplemental Figure S8, A and B), and found that the expression levels of MdPSY2-1 (MD09G1146800) and MdLCYb (MD06G1049200) were significantly higher in 'Beni Shogun' than in 'Yanfu 3' at 60, 30 and 0 DBFR (Figure 1, and and Supplemental Figure S8B). In addition, the MdPSY2-1 expression level was positively correlated with phytoene content and MdMYBS1 expression level, and the MdLCYb expression level was positively correlated with -carotene content and MdMYBS1 expression level (Supplemental Figure S9A). Moreover, expression levels of MdPSY2-1 and MdLCYb were high in MdMYBS1-overexpressing apple fruits, calli and leaves, and low in MdMYBS1-silenced apple fruits and calli (Figure 4A). These results suggest MdMYBS1 接下来，我们推断 MdMYBS1 可能通过调节类胡萝卜素生物合成基因来调控类胡萝卜素的积累。我们在 RNA-seq 数据中识别了类胡萝卜素生物合成基因（补充图 S8，A 和 B），发现在 60、30 和 0 DBFR 时，'贝尼将军'中 MdPSY2-1（MD09G1146800）和 MdLCYb（MD06G1049200）的表达水平显著高于'盐阜 3 号'（图 1， 和 以及补充图 S8B）。此外，MdPSY2-1 的表达水平与植物油脂含量和 MdMYBS1 的表达水平呈正相关，MdLCYb 的表达水平与 -胡萝卜素含量和 MdMYBS1 的表达水平呈正相关（补充图 S9A）。此外，MdPSY2-1 和 MdLCYb 在表达 MdMYBS1 的苹果果实、胼胝体和叶片中的表达水平较高，而在沉默 MdMYBS1 的苹果果实和胼胝体中的表达水平较低（图 4A）。这些结果表明
might influence carotenoids biosynthesis by regulating MdPSY2-1 and MdLCYb. 可能通过调节 MdPSY2-1 和 MdLCYb 影响类胡萝卜素的生物合成。
Previous study demonstrated that overexpressing MdPSY2-1 increases levels of total carotenoid, phytoene, phytofluene and -carotene (Dang et al., 2021). Here, overexpressing MdLCYb in apple calli enhanced levels of -carotene, -cryptoxanthin, violaxanthin and neoxanthin, whereas it decreased phytoene content (Supplemental Figure S10). These results indicate that MdPSY2-1 and MdLCYb contribute to carotenoid biosynthesis. 先前的研究表明，过表达 MdPSY2-1 可提高类胡萝卜素总量、植物烯、植物芴和 - 胡萝卜素的水平（Dang 等人，2021 年）。在这里，在苹果胼胝体中过表达 MdLCYb 可提高 -胡萝卜素、 -隐黄素、小叶黄素和新黄素的含量，而降低植物烯的含量（补充图 S10）。这些结果表明，MdPSY2-1 和 MdLCYb 有助于类胡萝卜素的生物合成。
We predicted the cis-elements of MdMYBS1 using JASPAR 2020. Previous studies reported that several R1-type proteins could specifically bind to a core sequence (HY)TATC(YD) (Lu et al., 2002; Lu et al., 2014; Liu et al., 2022). The main cis-elements of 'ATTATCTT' (-1741 bp, S1), 'CTTATCGT' (-1613 bp, S2), 'TTTATCTA' (-865 bp, S3) and 'AACTATCT' (-760 bp, S4) were identified within the MdPSY2-1 promoter. The main cis-elements of 'CTTATCCA' ( ), 'GCTATCTA' (-735 bp, S2), 'AAATATCG' (-415 bp, S3) and 'TTTATCTG' (-108 bp, S4) were identified within the MdLCYb promoter (Figure 4B). Y1H assay confirmed that MdMYBS1 binds to the promoters of MdPSY2-1 and MdLCYb (Supplemental Figure S11). 我们使用 JASPAR 2020 预测了 MdMYBS1 的顺式元件。之前的研究报道了几种 R1 型蛋白可以特异性地与核心序列 (HY)TATC(YD) 结合（Lu 等人，2002；Lu 等人，2014；Liu 等人，2022）。在 MdPSY2-1 启动子中发现了'ATTATCTT'（-1741 bp，S1）、'CTTATCGT'（-1613 bp，S2）、'TTTATCTA'（-865 bp，S3）和'AACTATCT'（-760 bp， S4）的主要顺式元件。在 MdLCYb 启动子中发现了'CTTATCCA' ( )、'GCTATCTA'（-735 bp，S2）、'AAATATCG'（-415 bp，S3）和'TTTATCTG'（-108 bp，S4）等主要顺式元件（图 4B）。Y1H 分析证实 MdMYBS1 与 MdPSY2-1 和 MdLCYb 的启动子结合（补充图 S11）。
Next, we confirmed the binding of MdMYBS1 to these major cis-elements in vivo using a chromatin immunoprecipitation (ChIP-PCR) assay. We obtained MdMYBS1-GFP fusion protein from MdMYBS1-overexpressing apple calli; PRI101-GFP (GFP) calli were used as a negative control. MdMYBS1 enhanced the PCR-based detection of gene promoters with the major motif TTATC(YD) or (HY)TATCCA, including the promoters of MdPSY2-1 and MdLCYb (Figure 4B). In addition, we found that MdMYBS1 directly bound to the TTTATCTA motif of the MdPSY2-1 接下来，我们使用染色质免疫沉淀（ChIP-PCR）检测法证实了 MdMYBS1 与这些主要顺式元件的体内结合。我们从MdMYBS1表达的苹果胼胝体中获得了MdMYBS1-GFP融合蛋白；PRI101-GFP（GFP）胼胝体作为阴性对照。MdMYBS1 增强了基于 PCR 的对具有主要基序 TTATC(YD) 或 (HY)TATCCA 的基因启动子的检测，包括 MdPSY2-1 和 MdLCYb 的启动子（图 4B）。此外，我们还发现 MdMYBS1 与 MdPSY2-1 启动子的 TTTATCTA 主题直接结合（图 4B）。
promoter or the CTTATCCA motif of the MdLCYb promoter using an electrophoretic mobility shift assay (EMSA) (Figure 4C). Furthermore, we explored how MdMYBS1 influences promoter activity in vivo using a luciferase (LUC) transactivation assay. MdMYBS1 can significantly facilitate the promoter activities of MdPSY2-1 and MdLCYb (Figure 4D). These results suggest MdMYBS1 acts as a transcriptional activator of MdPSY2-1 and MdLCYb by directly binding to their promoters to regulate carotenoid biosynthesis. 图 4C）。此外，我们还利用荧光素酶（LUC）转录激活试验探讨了 MdMYBS1 如何影响体内启动子的活性。MdMYBS1 能显著促进 MdPSY2-1 和 MdLCYb 的启动子活性（图 4D）。这些结果表明，MdMYBS1 可作为 MdPSY2-1 和 MdLCYb 的转录激活因子，直接与它们的启动子结合，从而调控类胡萝卜素的生物合成。
MdMYBS1 regulates ABA accumulation by activating MdNCED1 MdMYBS1 通过激活 MdNCED1 来调节 ABA 的积累
We reasoned that MdMYBS1 might regulate ABA accumulation by modulating ABA biosynthetic genes. We identified ABA biosynthesis genes in RNA-seq data (Supplemental Figure S8, A and C), and showed that the MdNCED1 (MD05G1282700) expression level was significantly higher in 'Beni Shogun' than in 'Yanfu 3' at 60, 30 and 0 DBFR (Figure 1L). In addition, the MdNCED1 expression level was positively correlated with ABA content and MAMYBS1 expression level (Supplemental Figure S9B). Moreover, the MdNCED1 expression level was high in MdMYBS1-overexpressing apple fruits, calli and leaves, and was low in MdMYBS1-silenced apple fruits and calli (Figure 5A). These results suggest MdMYBS1 might influence ABA accumulation by regulating MdNCED1. 我们推断 MdMYBS1 可能通过调节 ABA 生物合成基因来调控 ABA 的积累。我们在 RNA-seq 数据中鉴定了 ABA 生物合成基因（补图 S8，A 和 C），结果表明在 60、30 和 0 DBFR 时，'贝尼将军'中 MdNCED1（MD05G1282700）的表达水平显著高于'盐阜 3 号'（图 1L）。此外，MdNCED1 表达水平与 ABA 含量和 MAMYBS1 表达水平呈正相关（补图 S9B）。此外，在 MdMYBS1 表达的苹果果实、胼胝体和叶片中，MdNCED1 表达水平较高，而在 MdMYBS1 沉默的苹果果实和胼胝体中，MdNCED1 表达水平较低（图 5A）。这些结果表明 MdMYBS1 可能通过调节 MdNCED1 影响 ABA 的积累。
Here, we overexpressed or silenced MdNCED1 in apple calli (Figure 5, B and C). The ABA level was much higher, and levels of neoxanthin, violaxanthin, -cryptoxanthin and -carotene were much lower in MdNCED1-overexpressing calli, while silencing of MdNCED1 had the opposite effect (Figure 5, D and E). These results indicate that MdNCED1 contributes to ABA accumulation. 在这里，我们在苹果胼胝体中过表达或沉默了 MdNCED1（图 5，B 和 C）。在过表达 MdNCED1 的胼胝体中，ABA 水平更高，新黄素、中黄素、 -隐黄素和 -胡萝卜素的水平更低，而沉默 MdNCED1 则效果相反（图 5，D 和 E）。这些结果表明，MdNCED1 有助于 ABA 的积累。
The main cis-elements bound by MdMYBS1 of 'ACTATCCA' (-1794 bp, S1), 'AAGTATCT' (-1637 bp, S2), 'ATATATCT' (-1317 bp, S3) and 'ATTATCCT' (-1033 bp, S4) were identified within the MdNCED1 promoter. Y1H assay confirmed that MdMYBS1 binds to the MdNCED1 promoter (Supplemental Figure S11). Next, we confirmed the binding of MdMYBS1 to these major cis-elements using a ChIP-PCR assay. MdMYBS1 enhanced the PCR-based detection of MdNCED1 promoter with the major motif TTATC(YD) or (HY)TATCCA (Figure 5F). In addition, we found that MdMYBS1 directly bound to the ATTATCCT motif of MdNCED1 promoter using an EMSA (Figure 5G). Furthermore, we found that MdMYBS1 can significantly facilitate MdNCED1 promoter activity using a LUC transactivation assay (Figure 5H). These results suggest MdMYBS1 acts as a transcriptional activator of MdNCED1 by directly binding to its promoter to enhance ABA accumulation. 在 MdNCED1 启动子中发现了与 MdMYBS1 结合的主要顺式元件：'ACTATCCA'（-1794 bp，S1）、'AAGTATCT'（-1637 bp，S2）、'ATATATCT'（-1317 bp，S3）和'ATTATCCT'（-1033 bp，S4）。Y1H 分析证实 MdMYBS1 与 MdNCED1 启动子结合（补充图 S11）。接下来，我们用 ChIP-PCR 检测证实了 MdMYBS1 与这些主要顺式元件的结合。MdMYBS1 增强了基于 PCR 的 MdNCED1 启动子与主要图案 TTATC(YD) 或 (HY)TATCCA 的检测（图 5F）。此外，我们还通过 EMSA 发现 MdMYBS1 与 MdNCED1 启动子的 ATTATCCT 基序直接结合（图 5G）。此外，我们还利用 LUC 转录激活试验发现，MdMYBS1 能显著促进 MdNCED1 启动子的活性（图 5H）。这些结果表明，MdMYBS1 作为 MdNCED1 的转录激活剂，可直接与其启动子结合，从而增强 ABA 的积累。
The MdMYBS1 promoter was activated by exogenous ABA and MdABI5 外源 ABA 和 MdABI5 激活了 MdMYBS1 启动子。
We identified three cis-acting ABA-responsive element (ABRE) motifs in the MdMYBS1 promoter (S1: -1978 bp, CCACGTGG; S2: , CCACGTGG; S3: , TACGTG). We reasoned that acts on these motifs to regulate MdMYBS1 expression. To examine the impact of ABA on MdMYBS1 transcription, we generated ProMdMYBS1:LUC/GUS fusion constructs containing the 2016-bp promoter sequence of MdMYBS1 fused to the LUC or GUS reporter. When we expressed the ProMdMYBS1:LUC construct in Nicotiana benthamiana leaves, LUC activity significantly increased under ABA treatment compared to in the control ( treatment) (Figure 6A). Furthermore, we transformed apple calli with the ProMdMYBS1:GUS construct. Significantly higher GUS activity was detected in transgenic calli under ABA treatment than in the control (Figure 6B). These results suggest exogenous ABA promotes 13 我们在 MdMYBS1 启动子中发现了三个顺式作用的 ABA 响应元件（ABRE）基团（S1: -1978 bp, CCACGTGG; S2: , CCACGTGG; S3: , TACGTG）。我们推断 作用于这些基序来调控 MdMYBS1 的表达。为了研究 ABA 对 MdMYBS1 转录的影响，我们生成了 ProMdMYBS1:LUC/GUS 融合构建体，该构建体包含与 LUC 或 GUS 报告基因融合的 2016 bp MdMYBS1 启动子序列。当我们在烟曲霉叶片中表达 ProMdMYBS1:LUC 构建体时，与对照（ ）相比，在 ABA 处理下 LUC 活性显著增加（图 6A）。此外，我们用 ProMdMYBS1:GUS 构建物转化了苹果胼胝体。 ABA 处理下的转基因胼胝体中检测到的 GUS 活性明显高于对照（图 6B）。这些结果表明外源 ABA 促进 13
MdMYBS1 transcription by enhancing its promoter activity. 通过增强 MdMYBS1 启动子的活性来实现其转录。
Next, we searched for the TFs that specifically bind to ABRE motif in the MdMYBS1 promoter. The ABRE/G-box motif is recognized by ABI5 TFs (Zinsmeister et al., 2016; Zhao et al., 2020; Song et al., 2022). Using Y1H screening (Jia et al., 2021) and RNA-seq data, one MdABI5 (MD14G1021600) was identified. MdABI5 transcript level was significantly higher in 'Beni Shogun' than in 'Yanfu 3' at 60, 30 and 0 DBFR (Supplemental Figure S12A), and was correlated with total carotenoid and ABA content (Supplemental Figure S12, B and C). Y1H assay validated that MdABI5 binds the MdMYBS1 promoter (Figure ). 接下来，我们搜索了能与 MdMYBS1 启动子中 ABRE 基因组特异性结合的因子。ABRE/G-box马达被ABI5 TF识别（Zinsmeister等人，2016；赵等人，2020；宋等人，2022）。通过 Y1H 筛选（Jia 等人，2021 年）和 RNA-seq 数据，发现了一个 MdABI5（MD14G1021600）。在 60、30 和 0 DBFR 时，'贝尼将军'的 MdABI5 转录水平明显高于'盐阜 3 号'（补充图 S12A），并且与类胡萝卜素和 ABA 的总含量相关（补充图 S12，B 和 C）。Y1H 分析验证了 MdABI5 与 MdMYBS1 启动子的结合（图 ）。
Next, we confirmed the binding of MdABI5 to the ABRE motif in MdMYBS1 promoter using a ChIP-PCR assay. We obtained the MdABI5-GFP fusion protein from MdABI5-overexpressing apple calli; P101-GFP calli were used as a negative control. MdABI5 heightened the PCR-based detection of the MdMYBS1 promoter with ABRE/G-box motifs (Figure 6D), and EMSA demonstrated MdABI5 bound to the TACGTG motif of ABRE/G-box in the MdMYBS1 promoter (Figure 6E). Furthermore, we found that MdABI5 can significantly facilitate the MdMYBS1 promoter activity using a LUC transactivation assay (Figure 6F) and a GUS reporter assay (Figure 6G). These results suggest MdABI5 acts as a transcriptional activator of MdMYBS1 by directly binding to its promoter. 接着，我们用 ChIP-PCR 检测法证实了 MdABI5 与 MdMYBS1 启动子中的 ABRE 基序的结合。我们从MdABI5-overexpressing苹果胼胝体中获得了MdABI5-GFP融合蛋白；P101-GFP胼胝体作为阴性对照。MdABI5 提高了基于 PCR 的 MdMYBS1 启动子与 ABRE/G-box 基序的检测（图 6D），EMSA 表明 MdABI5 与 MdMYBS1 启动子中 ABRE/G-box 的 TACGTG 基序结合（图 6E）。此外，我们还利用 LUC 反式激活试验（图 6F）和 GUS 报告试验（图 6G）发现，MdABI5 能显著促进 MdMYBS1 启动子的活性。这些结果表明，MdABI5 通过直接与 MdMYBS1 启动子结合，起到了转录激活剂的作用。
MdABI5 regulates carotenoids and accumulation, and responds to MdABI5 可调节类胡萝卜素和 的积累，并对以下因素做出反应
To investigate the role of MdABI5 in carotenoid biosynthesis, we separately transiently transformed 'Granny Smith' fruit with the 35S:MdABI5 (MdABI5-OVX) and TRV:MdABI5 14 为了研究 MdABI5 在类胡萝卜素生物合成中的作用，我们分别用 35S:MdABI5（MdABI5-OVX）和 TRV:MdABI5 14 对'Granny Smith'果实进行了瞬时转化。
(MdABI5-TRV) constructs (Figure 7, A and B). Deep yellow coloration was observed in MdABI5-OVX fruit skin (Figure 7B). Levels of total carotenoid and endogenous ABA were markedly higher in MdABI5-OVX fruit skin yet lower in MdABI5-TRV fruit skin vs. the in controls (P101F and TRV, respectively) (Figure 7, C and D). Furthermore, MdABI5, MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1 were expressed at much higher levels in MdABI5-OVX fruit skin yet at much lower levels in MdABI5-RNAi fruit skin than in the controls (Figure 7E). These findings suggest that MdABI5 promotes carotenoid and endogenous ABA accumulation. (图 7，A 和 B）。在 MdABI5-OVX 果皮中观察到深黄色着色（图 7B）。与对照组（分别为 P101F 和 TRV）相比，MdABI5-OVX 果皮的类胡萝卜素总量和内源 ABA 水平明显较高，而 MdABI5-TRV 果皮则较低（图 7，C 和 D）。此外，MdABI5、MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1 在 MdABI5-OVX 果皮中的表达水平远高于对照组，但在 MdABI5-RNAi 果皮中的表达水平远低于对照组（图 7E）。这些发现表明，MdABI5 可促进类胡萝卜素和内源 ABA 的积累。
MdABI5 was also overexpressed (MdABI5-OVX) or silenced (MdABI5-RNAi) in apple calli (Figure 7, A and F). Significantly higher levels of total carotenoid and endogenous ABA were detected in MdABI5-OVX calli yet lower in MdABI5-RNAi calli vs. the controls (Figure 7, G and H). In addition, overexpressing MdABI5 upregulated MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1, whereas silencing MdABI5 had the opposite effect (Figure 7I). Taken together, these findings suggest that MdABI5 promotes carotenoid and endogenous ABA accumulation by activating MdMYBS1 transcription. MdABI5 也在苹果胼胝体中过表达（MdABI5-OVX）或沉默（MdABI5-RNAi）（图 7，A 和 F）。与对照组相比，在 MdABI5-OVX 苹果胼胝体中检测到的类胡萝卜素总量和内源 ABA 水平明显较高，而在 MdABI5-RNAi 苹果胼胝体中则较低（图 7，G 和 H）。此外，过表达 MdABI5 会上调 MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1，而沉默 MdABI5 则产生相反的效果（图 7I）。综上所述，这些发现表明 MdABI5 通过激活 MdMYBS1 的转录促进类胡萝卜素和内源 ABA 的积累。
Next, we amplified the MdABI5 promoter ( ) and identified several ABRE motifs within it. We reasoned that ABA acts on the ABRE motifs to regulate MAABI5 expression. When we transiently transformed . benthamiana leaves with the ProMdABI5:LUC fusion construct, LUC activity increased significantly under ABA treatment compared to in the control ( treatment) (Figure 7J). Moreover, we transformed apple calli with the ProMdABI5:GUS fusion construct. GUS activity was significantly higher in transgenic calli under treatment compared to in the control (Figure 7K). These results demonstrate that MdABI5 could be 15 接着，我们扩增了 MdABI5 启动子 ( ) 并确定了其中的几个 ABRE 基序。我们推断 ABA 作用于 ABRE 基序来调节 MAABI5 的表达。当我们用 ProMdABI5:LUC 融合构建体瞬时转化 .benthamiana 叶片时，与对照组相比（ 处理），在 ABA 处理下 LUC 活性显著增加（图 7J）。此外，我们用 ProMdABI5:GUS 融合构建体转化了苹果胼胝体。在 处理下，转基因胼胝体的 GUS 活性明显高于对照（图 7K）。这些结果表明，MdABI5 可 15
activated by exogenous ABA. 被外源 ABA 激活。
MdABI5-MdMYBS1 cascade regulates carotenoid and ABA accumulation MdABI5-MdMYBS1 级联调节类胡萝卜素和 ABA 的积累
We explored how the MdABI5-MdMYBS1 transcriptional cascade influences carotenoid biosynthesis by silencing MdMYBS1 (MdMYBS1-RNAi) in apple calli overexpressing MdABI5 (MdABI5-OVX) and silencing MdABI5 (MdABI5-RNAi) in apple calli overexpressing MdMYBS1 (MdMYBS1-OVX) (Figure 8A). MdMYBS1 was significantly silenced in MdABI5-OVX/MdMYBS1-RNAi calli, and MdABI5 was significantly silenced in MdMYBS1-OVX/MdABI5-RNAi calli (Figure 8B). The silencing of MdMYBS1 significantly suppressed total carotenoid and ABA production in MdABI5-OVX calli, however, total carotenoid and ABA levels were not significantly affected when MdABI5 was silenced in MdMYBS1-OVX calli (Figure 8, C and D). Furthermore, the silencing of MdMYBS1 significantly suppressed expression levels of MdPSY2-1, MdLCYb and MdNCED1 in MdABI5-OVX calli, however, these were not significantly affected when MdABI5 was silenced in MdMYBS1-OVX calli (Figure 8E). These results demonstrate that MdABI5 regulates the transcription of MdMYBS1, and MdMYBS1 activates MdPSY2-1, MdLCYb and MdNCED1 expression. Thus, the MdABI5-MdMYBS1 cascade regulates carotenoid biosynthesis and ABA accumulation. 我们通过沉默过表达 MdABI5（MdABI5-OVX）的苹果胼胝体中的 MdMYBS1（MdMYBS1-RNAi）和沉默过表达 MdMYBS1（MdMYBS1-OVX）的苹果胼胝体中的 MdABI5（MdABI5-RNAi），探索了 MdABI5-MdMYBS1 转录级联如何影响类胡萝卜素的生物合成（图 8A）。在 MdABI5-OVX/MdMYBS1-RNAi 胼胝体中，MdMYBS1 被显著沉默；在 MdMYBS1-OVX/MdABI5-RNAi 胼胝体中，MdABI5 被显著沉默（图 8B）。沉默 MdMYBS1 能显著抑制 MdABI5-OVX 胼胝体中类胡萝卜素和 ABA 的总产生量，但沉默 MdMYBS1-OVX 胼胝体中的 MdABI5 对类胡萝卜素和 ABA 的总产生量没有显著影响（图 8，C 和 D）。此外，在 MdABI5-OVX 胼胝体中，沉默 MdMYBS1 能显著抑制 MdPSY2-1、MdLCYb 和 MdNCED1 的表达水平，但在 MdMYBS1-OVX 胼胝体中沉默 MdABI5 时，这些表达水平没有受到显著影响（图 8E）。这些结果表明，MdABI5 可调控 MdMYBS1 的转录，而 MdMYBS1 可激活 MdPSY2-1、MdLCYb 和 MdNCED1 的表达。因此，MdABI5-MdMYBS1 级联调控类胡萝卜素的生物合成和 ABA 的积累。
Exogenous ABA promotes carotenoid accumulation 外源 ABA 可促进类胡萝卜素的积累
ABA can promote carotenoid accumulation in tomato and citrus fruits (Wu et al., 2018; Sun et al., 2023). In this study, ABA content was markedly higher in 'Beni Shogun' than in 'Yanfu 3' during fruit ripening (Figure ), and MdMYBS1 activated MdNCED1 expression to promote ABA 16 ABA 可以促进番茄和柑橘类水果中类胡萝卜素的积累（Wu 等，2018；Sun 等，2023）。在本研究中，果实成熟过程中'贝尼将军'的 ABA 含量明显高于'烟富 3 号'（图 ），MdMYBS1 激活了 MdNCED1 的表达以促进 ABA 16
accumulation (Figure 5). We reasoned that ABA could influence carotenoid accumulation in apple fruit. To decipher the potential relationship between ABA and carotenoid accumulation in apple, we harvested 'Orin' fruits and treated them with exogenous ABA ( , or via vacuum injection, and we also vacuum-injected fruits with the ABA inhibitor NDGA ( ). Treatment with 0.5 or ABA accelerated yellow color development in apple fruit, whereas treatment with NDGA suppressed yellow color development (Figure 9A). ABA treatment strongly induced accumulation of ABA and total carotenoid in fruit flesh, whereas these levels were much lower in fruit flesh under NDGA treatment compared to in controls (Figure 9B, C). MdAB15, MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1 were markedly upregulated in fruit flesh treated with 0.5 or ABA (Figure 9D). These results indicate that exogenous ABA promotes carotenoid production by enhancing the expression of carotenoid-related TF genes and carotenoid biosynthetic genes. 图 5）。我们推断 ABA 可能会影响苹果果实中类胡萝卜素的积累。为了弄清 ABA 与类胡萝卜素在苹果中积累的潜在关系，我们采收了'Orin'果实，用外源 ABA ( , 或 通过真空注射处理，我们还用 ABA 抑制剂 NDGA ( ) 真空注射处理果实。用 0.5 或 ABA 处理会加速苹果果实的黄颜色发展，而用 NDGA 处理则会抑制黄颜色发展（图 9A）。ABA 处理强烈诱导果肉中 ABA 和总类胡萝卜素的积累，而 NDGA 处理下果肉中的这些含量比对照组低得多（图 9B、C）。在用 0.5 或 ABA 处理的果肉中，MdAB15、MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1 被明显上调（图 9D）。这些结果表明，外源 ABA 可通过提高类胡萝卜素相关 TF 基因和类胡萝卜素生物合成基因的表达来促进类胡萝卜素的生成。
Discussion 讨论
Carotenoid is an important pigment for fruit coloration with considerable nutritional value and has become a major target trait in apple breeding (Ampomah-Dwamena et al., 2012, 2022; Dang et al., 2021). Carotenoid content and compounds display substantial variability in different Malus genotypes. In the current study, we identified carotenoid as the main factor influencing apple flesh color in 'Red Fuji' bud mutants 'Beni Shogun' and 'Yanfu 3' (Figure 1 and Supplemental Figure S1). Among carotenoid compounds, lutein content decreases whereas phytoene and -carotene contents increase during fruit ripening in several plants such as carrot (Daucus carota ssp. sativus) (Clotault et al., 2008), orange (Guzman et al., 2010), and apple (Ampomah-Dwamena 17 类胡萝卜素是果实着色的重要色素，具有相当高的营养价值，已成为苹果育种的主要目标性状（Ampomah-Dwamena 等人，2012 年，2022 年；Dang 等人，2021 年）。类胡萝卜素的含量和化合物在不同的 Malus 基因型中存在很大差异。在本研究中，我们发现类胡萝卜素是影响'红富士'芽变体'贝尼将军'和'延富 3 号'苹果果肉颜色的主要因素（图 1 和补充图 S1）。在胡萝卜（Daucus carota ssp. sativus）（Clotault 等人，2008 年）、橘子（Guzman 等人，2010 年）和苹果（Ampomah-Dwamena 17）等几种植物的果实成熟过程中，类胡萝卜素含量减少，而植物色素和 - 胡萝卜素含量增加。
et al., 2012; Ampomah-Dwamena et al., 2022); we observed a similar pattern in 'Yanfu 3' and 'Beni Shogun' (Figure 1). The predominant compounds in ripening apple fruits are -carotene and -cryptoxanthin in 'Aotea', violaxanthin and neoxanthin in 'Royal Gala', and lutein in 'Granny Smith' (Ampomah-Dwamena et al., 2012). Here, we detected six carotenoid compounds in ripening apple fruits. The major compounds were -carotene and phytoene in 'Beni Shogun', -carotene and -cryptoxanthin in 'Yanfu 3', and lutein and -carotene in 'Granny Smith' (Figures 1 and 3). The different predominant compounds in different Malus genotypes might be attributed to differential transcriptional or post-transcriptional regulation (Dang et al., 2021; Ampomah-Dwamena et al., 2022). Analyzing the genetic variations in carotenoid biosynthesis could help to decipher the regulatory mechanism of carotenoid biosynthesis in apple fruit. Furthermore, multiple Malus genotypes with different carotenoid contents could be used to produce new apple varieties with high carotenoid contents. 等人，2012 年；Ampomah-Dwamena 等人，2022 年）；我们在'盐富 3 号'和'贝尼将军'中也观察到类似的模式（图 1）。成熟苹果果实中最主要的化合物是 -胡萝卜素和 -隐黄素（'Aotea'）、中黄素和新黄素（'Royal Gala'）以及叶黄素（'Granny Smith'）（Ampomah-Dwamena 等人，2012 年）。在这里，我们在成熟的苹果果实中检测到六种类胡萝卜素化合物。主要化合物有：'Beni Shogun'中的 -胡萝卜素和植物色素，'Yanfu 3'中的 -胡萝卜素和 -隐黄素，以及'Granny Smith'中的叶黄素和 -胡萝卜素（图 1 和图 3）。不同马蔺基因型的主要化合物不同，可能是由于转录或转录后调控不同所致（Dang 等人，2021 年；Ampomah-Dwamena 等人，2022 年）。分析类胡萝卜素生物合成的遗传变异有助于破译苹果果实中类胡萝卜素生物合成的调控机制。此外，还可利用类胡萝卜素含量不同的多种马蔺基因型来培育类胡萝卜素含量高的苹果新品种。
Carotenoids are isoprenoid compounds that are synthesized by metabolic enzymes (Yuan et al., 2015). The diversity of carotenoid pigmentation is influenced by carotenoid biosynthetic genes (Diretto et al., 2006; Jang et al., 2020; Sun et al., 2020; Zhang et al., 2020). Our previous research demonstrated overexpressing MdPSY2-1 increases the phytoene and total carotenoid contents in apple (Dang et al., 2021). Here, the MdPSY2-1 or MdLCYb expression were highly positively correlated with phytoene or -carotene content in apple fruits (Figure 1 and Supplemental Figure S9), and we determined that overexpressing MdLCYb enhanced levels of -carotene, -cryptoxanthin, violaxanthin and neoxanthin whereas it decreased phytoene content (Supplemental Figure S10), indicating that MdPSY2-1 and MdLCYb are major biosynthetic genes 18 类胡萝卜素是由代谢酶合成的异戊烯化合物（Yuan 等，2015 年）。类胡萝卜素色素的多样性受类胡萝卜素生物合成基因的影响（Diretto 等人，2006 年；Jang 等人，2020 年；Sun 等人，2020 年；Zhang 等人，2020 年）。我们之前的研究表明，过表达 MdPSY2-1 能增加苹果中植物烯和总类胡萝卜素的含量（Dang 等人，2021 年）。在这里，MdPSY2-1 或 MdLCYb 的表达与苹果果实中的植物烯或 - 胡萝卜素含量高度正相关（图 1 和补充图 S9），并且我们确定过表达 MdLCYb 提高了 - 胡萝卜素的水平、 我们确定，过表达 MdLCYb 可提高 -胡萝卜素、-隐黄素、中黄素和新黄素的含量，而降低植物色素含量（补充图 S10），这表明 MdPSY2-1 和 MdLCYb 是主要的生物合成基因 18
contributing to carotenoid biosynthesis in apple. 有助于苹果中类胡萝卜素的生物合成。
Previous studies reported that violaxanthin and neoxanthin are precursors of ABA (Perreau et al., 2020; Kavi Kishor et al., 2022). In the study, Neoxanthin content was much lower in 'Beni Shogun' fruit with higher levels of carotenoids and ABA than in 'Yanfu 3' fruit with lower levels of carotenoids and ABA (Figure 1). Moreover, the neoxanthin level highly declined in MdMYBS1-overexpressing apple fruits, calli and leaves, and highly increased in MdMYBS1-silenced apple fruits and calli (Figure 3). These results indicate that the neoxanthin is the main precursor of ABA synthesis. 以前的研究报告称，玉米黄质和新黄质是 ABA 的前体（Perreau 等人，2020 年；Kavi Kishor 等人，2022 年）。在本研究中，类胡萝卜素和 ABA 含量较高的 "贝尼将军 "果实中的新黄质含量远低于类胡萝卜素和 ABA 含量较低的 "盐阜 3 号 "果实（图 1）。此外，MdMYBS1 基因表达的苹果果实、胼胝体和叶片中的新黄质含量显著下降，而 MdMYBS1 基因沉默的苹果果实和胼胝体中的新黄质含量显著上升（图 3）。这些结果表明，新黄质是合成 ABA 的主要前体。
MYB TFs play vital roles in growth and development in diverse plants (Dubos et al., 2010; Wu et al., 2022). Several MYBs activate or repress carotenoid biosynthesis by directly modulating the transcription of carotenogenic genes (Sagawa et al., 2016; Meng et al., 2019; Wu et al., 2020). For example, CrMYB68 inhibits - and - branch carotenoid biosynthesis in citrus (Zhu et al., 2017), whereas AdMYB7 promotes carotenoid production by directly enhancing AdLCY-B expression in kiwifruit (Ampomah-Dwamena et al., 2019). Here, we found that MdMYBS1 contributes to fruit flesh color of apple and tomato (Figure 3). MdMYBS1 acts as a activator of the carotenogenic genes MdPSY2-1 and MdLCYb by directly binding to their promoters (Figure 4). These findings indicate that different subclasses of MYB TFs play multiple roles in modulating carotenoid biosynthesis via controlling the expression of different carotenogenic genes in diverse plant species. MYB TFs 在多种植物的生长和发育过程中发挥着重要作用（Dubos 等人，2010 年；Wu 等人，2022 年）。一些 MYB 通过直接调节类胡萝卜素生成基因的转录来激活或抑制类胡萝卜素的生物合成（Sagawa 等人，2016 年；Meng 等人，2019 年；Wu 等人，2020 年）。例如，CrMYB68 可抑制柑橘中 - 和 - 分支类胡萝卜素的生物合成（Zhu 等人，2017 年），而 AdMYB7 可通过直接增强猕猴桃中 AdLCY-B 的表达来促进类胡萝卜素的产生（Ampomah-Dwamena 等人，2019 年）。在这里，我们发现 MdMYBS1 对苹果和番茄的果肉颜色有促进作用（图 3）。MdMYBS1 作为胡萝卜素基因 MdPSY2-1 和 MdLCYb 的激活剂，直接与它们的启动子结合（图 4）。这些发现表明，在不同植物物种中，不同亚类的 MYB TFs 通过控制不同胡萝卜素生成基因的表达，在调节类胡萝卜素生物合成方面发挥着多重作用。
In perennial fruits, somatic mutations known as "bud sports" can arise from epigenetic or genetic 在多年生水果中，被称为 "花蕾运动 "的体细胞突变可能源于表观遗传或基因遗传
alterations (Kobayashi et al., 2004; El-Sharkawy et al., 2015; Jia et al., 2020; Li et al., 2020). Both 'Yanfu 3' and 'Beni Shogun' are bud mutants of Red Fuji. The MdMYBS1 transcript level was significantly higher in 'Beni Shogun' than in 'Yanfu 3' during fruit ripening (Figure 1), however, we could not find the genetic DNA variants of MdMYBS1 including promoter, exons and introns between 'Beni Shogun' and 'Yanfu 3' (Supplemental Table S2). Previous studies indicated that epigenetic regulation, such as methylation and histone acetylation, is involved in carotenoid synthesis (Arango et al., 2016; Li et al., 2022). We speculate that the epigenetic regulation of MAMYBS1 is involved in regulating carotenoid biosynthesis, however this requires further study. Kobayashi 等人，2004；El-Sharkawy 等人，2015；Jia 等人，2020；Li 等人，2020）。延富 3 号 "和 "贝尼将军 "都是红富士的芽突变体。在果实成熟过程中，'贝尼将军'的MdMYBS1转录水平明显高于'烟富3号'（图1），但我们未能发现'贝尼将军'和'烟富3号'之间MdMYBS1的DNA遗传变异，包括 启动子、外显子和内含子（补充表S2）。以往的研究表明，甲基化和组蛋白乙酰化等表观遗传调控参与了类胡萝卜素的合成（Arango 等，2016；Li 等，2022）。我们推测 MAMYBS1 的表观遗传调控参与了类胡萝卜素生物合成的调控，但这还需要进一步研究。
The phytohormone ABA plays key roles in fruit development, coloration, and ripening (Leng et al., 2014). ABA is derived from the cleavage of carotenoid precursors. ABA biosynthesis is restricted by one rate-limiting step catalyzed by the enzyme NCED (Frey et al., 2012; Sato et al., 2018; Lang et al., 2021; Wang et al., 2021). Silencing SINCED1 reduces endogenous ABA levels, and increases contents of lycopene and -carotene in tomato fruits (Sun et al., 2012a, b). In our study, MdNCED1 was positively related to ABA accumulation during the ripening of apple fruit flesh (Figure 1 and Supplemental Figure S9), indicating that MdNCED1 is related to ABA accumulation during the ripening of apple fruit flesh. Moreover, overexpressing MdNCED1 in apple calli leads to a higher level and lower levels of neoxanthin, violaxanthin, -cryptoxanthin and -carotene (Figure 5), indicating that MdNCED1 is the major protein contributing to ABA accumulation. 植物激素 ABA 在果实发育、着色和成熟过程中发挥着关键作用（Leng 等人，2014 年）。ABA 来自类胡萝卜素前体的裂解。ABA 的生物合成受到 NCED 酶催化的一个限速步骤的限制（Frey 等人，2012 年；Sato 等人，2018 年；Lang 等人，2021 年；Wang 等人，2021 年）。沉默 SINCED1 会降低内源 ABA 水平，增加番茄果实中番茄红素和 - 胡萝卜素的含量（Sun 等，2012a, b）。在我们的研究中，MdNCED1 与苹果果肉成熟过程中的 ABA 积累呈正相关（图 1 和补充图 S9），表明 MdNCED1 与苹果果肉成熟过程中的 ABA 积累有关。此外，在苹果胼胝体中过表达 MdNCED1 会导致 水平升高，而新黄素、暴黄素、 -隐黄素和 -胡萝卜素的水平降低（图 5），表明 MdNCED1 是促进 ABA 积累的主要蛋白。
NCED gene expression is regulated by a series of TFs (Ma et al., 2018; Lang et al., 2021; Martín-Pizarro et al., 2021). Ethylene response transcription factor PpERF3 modulates ABA biosynthesis by enhancing PpNCED2/3 expression in peach (Wang et al., 2019). Here, MdNCED1 20 NCED 基因的表达受一系列 TFs 的调控（Ma 等人，2018 年；Lang 等人，2021 年；Martín-Pizarro 等人，2021 年）。乙烯响应转录因子 PpERF3 通过增强桃中 PpNCED2/3 的表达来调节 ABA 的生物合成（Wang 等，2019）。在这里，MdNCED1 20
expression and ABA levels were much higher in MAMYBS1-overexpressing fruits and calli and much lower in MdMYBS1-silenced fruits and calli (Figure 5). In addition, we demonstrated that MdMYBS1 enhances MdNCED1 expression via binding to its promoter (Figure 5), indicating that MdMYBS1 promotes ABA accumulation via activating MdNCED1 expression. 表达和 ABA 水平在 MAMYBS1 基因缺失的果实和胼胝体中要高得多，而在 MdMYBS1 基因沉默的果实和胼胝体中要低得多（图 5）。此外，我们还证明 MdMYBS1 通过与其启动子结合增强了 MdNCED1 的表达（图 5），表明 MdMYBS1 通过激活 MdNCED1 的表达促进了 ABA 的积累。
Exogenous ABA treatment induces fruit ripening in grape (Vitis vinifera) berries (Giribald et al., 2010). The interaction between ABA and ethylene modulates fig (Ficus carica) fruit ripening (Qiao et al., 2021). Apple fruit ripening is promoted by ethylene. However, apple fruit ripening is also regulated by the dynamic interaction between phytohormones (e.g., ethylene and ABA) (Ji et al., 2021). Both 'Yanfu 3' and 'Beni Shogun' are bud mutants of Red Fuji. However, 'Beni Shogun' fruits ripened one month earlier than 'Yanfu 3' fruits, and ABA content was significantly higher in 'Beni Shogun' fruit flesh compared to in 'Yanfu 3' (Figure 1). We propose that the higher endogenous content induces fruit ripening in 'Beni Shogun' and that the MdMYBS1-MdNCED1 module or MdMYBS1-interaction proteins are involved in regulating the early maturity of 'Beni Shogun' fruit; these ideas warrant further study. 外源 ABA 处理可诱导葡萄（Vitis vinifera）浆果成熟（Giribald 等人，2010 年）。ABA 和乙烯之间的相互作用可调节无花果（Ficus carica）果实的成熟（Qiao 等人，2021 年）。乙烯能促进苹果果实成熟。然而，苹果果实的成熟还受到植物激素（如乙烯和 ABA）之间动态相互作用的调控（Ji 等人，2021 年）。延富 3 号 "和 "贝尼将军 "都是红富士的花蕾突变体。然而，'贝尼将军'的果实比'延富 3 号'早熟一个月，而且'贝尼将军'果肉中的 ABA 含量明显高于'延富 3 号'（图 1）。我们认为，较高的内源 含量诱导了'贝尼将军'果实的成熟，而 MdMYBS1-MdNCED1 模块或 MdMYBS1 相互作用蛋白参与了'贝尼将军'果实早熟的调控；这些观点值得进一步研究。
ABRE motifs are present in numerous genes and respond to ABA (Fujita et al., 2005; Yoshida et al., 2010; Liu et al., 2022). We determined that ABA acts on ABRE motifs in the MdMYBS1 and MdABI5 promoters to influence their expressions (Figures 6 and 7). Therefore, MdMYBS1 and MdABI5 promotes ABA accumulation; in turn, higher ABA levels enhance MdMYBS1 and MdABI5 expression to promote carotenoid accumulation. The ABRE/G-box motif is recognized by ABI5 TFs (Zinsmeister et al., 2016; Zhao et al., 2020; Song et al., 2022). In this study, we identified MdABI5 as a transcriptional activator that specifically binds to the ABRE motif in the MdMYBS1 promoter 21 ABRE 基序存在于许多基因中，并对 ABA 有反应（Fujita 等人，2005 年；Yoshida 等人，2010 年；Liu 等人，2022 年）。我们确定 ABA 作用于 MdMYBS1 和 MdABI5 启动子中的 ABRE 基序，从而影响它们的表达（图 6 和图 7）。因此，MdMYBS1 和 MdABI5 可促进 ABA 的积累；反过来，较高的 ABA 水平可提高 MdMYBS1 和 MdABI5 的表达，从而促进类胡萝卜素的积累。ABRE/G-box motif 可被 ABI5 TFs 识别（Zinsmeister 等人，2016 年；Zhao 等人，2020 年；Song 等人，2022 年）。在本研究中，我们发现 MdABI5 是一种转录激活因子，可特异性地与 MdMYBS1 启动子中的 ABRE 矩阵结合 21
and enhances its transcription (Figure 6). ABI5 is a bZIP TF whose disruption increases carotenoid and chlorophyll biosynthesis in the green alga (Bai et al., 2021). Here, we demonstrated that MdABI5 responds to ABA and activates carotenoid accumulation (Figure 7). These findings suggest that additional TFs might be involved in ABA-regulated carotenoid biosynthesis, a notion that requires further study. 并增强其转录（图 6）。ABI5 是一种 bZIP TF，破坏它可增加绿藻中类胡萝卜素和叶绿素的生物合成（Bai 等人，2021 年）。在这里，我们证明 MdABI5 对 ABA 有反应并激活类胡萝卜素的积累（图 7）。这些发现表明，其他 TFs 可能参与了 ABA 调控的类胡萝卜素生物合成，这一观点需要进一步研究。
ABA influences carotenoid biosynthesis in diverse plants (Khaleghnezhad et al., 2019; Liu et al., 2020). For example, ABA promotes the accumulation of carotenoid during fruit color development in tomato (Barickman et al., 2014; Wu et al., 2018). The CsHB5-CsbZIP44 module regulates ABA-induced carotenoid biosynthesis in citrus (Sun et al., 2023). Here, we established that ABA treatment strongly induced carotenoid accumulation in apple fruit, and MAMYBS1 and MdABI5 were upregulated in ABA-treated fruits (Figure 9). These results confirm that promotes carotenoid production by enhancing the transcription of genes encoding carotenoid-related TFs. ABA 影响多种植物中类胡萝卜素的生物合成（Khaleghnezhad 等人，2019 年；Liu 等人，2020 年）。例如，在番茄果实颜色发育过程中，ABA 会促进类胡萝卜素的积累（Barickman 等人，2014 年；Wu 等人，2018 年）。CsHB5-CsbZIP44模块调控柑橘中ABA诱导的类胡萝卜素生物合成（Sun等人，2023年）。在这里，我们证实 ABA 处理可强烈诱导苹果果实中类胡萝卜素的积累，并且 MAMYBS1 和 MdABI5 在 ABA 处理的果实中上调（图 9）。这些结果证实 可通过增强类胡萝卜素相关 TFs 编码基因的转录来促进类胡萝卜素的产生。
In summary, MdMYBS1 contributes to carotenoid accumulation via activating carotenogenic genes MdPSY2-1 and MdLCYb. MdMYBS1 enhances ABA accumulation by activating MdNCED1 expression. In addition, MdABI5 activates MdMYBS1 expression by binding its promoter to increase carotenoid and ABA accumulation. Furthermore, ABA promotes carotenoid biosynthesis and enhances the MdMYBS1 and MdABI5 promoter activities (Figure 10). Overall, our findings promise to be a major advance in the regulatory mechanism that the MdABI5-MdMYBS1 cascade activated by regulates carotenoids and production, and lead the way to other avenues in the breeding and planting for genetic improvement of fruit coloration and quality. 22 总之，MdMYBS1 通过激活类胡萝卜素生成基因 MdPSY2-1 和 MdLCYb 来促进类胡萝卜素的积累。MdMYBS1 通过激活 MdNCED1 的表达来增强 ABA 的积累。此外，MdABI5 通过结合 MdMYBS1 的启动子激活其表达，从而增加类胡萝卜素和 ABA 的积累。此外，ABA 促进类胡萝卜素的生物合成，并增强 MdMYBS1 和 MdABI5 的启动子活性（图 10）。总之，我们的发现有望成为 激活的 MdABI5-MdMYBS1 级联调控类胡萝卜素和 生成的调控机制方面的重大进展，并为遗传改良果实着色和品质的育种和种植开辟其他途径。22
'Yanfu 3' is a red skin mutant and 'Beni Shogun' is an early mature mutant of 'Red Fuji' apple (Malus domestica Borkh.). 'Yanfu 3' and 'Beni Shogun' apple fruits were harvested and collected from the Laixi breeding farm (Laixi City, Qingdao City, Shandong Province, China) in 2019. The apple trees were grown under identical conditions using standard cultivation and management practices. Fruits were harvested every beginning at 60 days before fruit ripening (DBFR) and ending with mature fruit at 0 DBFR (Beni Shogun, September 28, 2019; Yanfu 3, October 28, 2019). At each harvesting time, 18 fruits were collected from eight trees and were divided into three sets, each containing six fruits; each set was considered to be one biological replicate. The fruit flesh of each set was collected and frozen in liquid nitrogen, and stored at to analyze carotenoid contents, plant hormone contents, and gene expression. 'Orin' calli were incubated at under dark conditions (Dang et al., 2021). 'Micro-Tom' tomato (Solanum lycopersicum) and Nicotiana benthamiana plantlets were incubated under a 16 -h-light/8-h-dark cycle at (Jia et 462 al., 2021; Zhu et al., 2023). '烟富3号'是红皮突变体，'贝尼将军'是'红富士'苹果（Malus domestica Borkh.）的早熟突变体。'烟富3号'和'贝尼将军'苹果果实于2019年从莱西育种场（中国山东省青岛市莱西市）采收并收集。苹果树在相同的条件下生长，采用标准的栽培和管理方法。果实从果实成熟前 60 天（DBFR）开始，到成熟果实为 0 DBFR 时结束（贝尼将军，2019 年 9 月 28 日；烟富 3 号，2019 年 10 月 28 日），每 。在每个采收期，从 8 棵树上采集 18 个果实，分成 3 组，每组 6 个果实；每组被视为一个生物重复。收集每组果实的果肉并用液氮冷冻，然后储存在 ，以分析类胡萝卜素含量、植物激素含量和基因表达。'Orin'茧在 黑暗条件下培养（Dang 等人，2021 年）。'Micro-Tom'番茄（Solanum lycopersicum）和烟草小株在 16 小时-光照/8 小时-黑暗循环条件下培养， （Jia et 462 al.，2021；Zhu et al.，2023）。
Exogenous ABA treatment in 'Orin' fruits 对'Orin'果实进行外源 ABA 处理
'Orin' fruits were harvested at 130 days after full blossom (DAFB) from the Laixi Gaosong farm 莱西高松农场的'Orin'果实在盛花后 130 天（DAFB）采收
vacuum-injected with , or ABA (catalog no. 862169 , Sigma-Aldrich, USA). For ABA inhibitor treatments, the fruits were vacuum-injected with NDGA (nordihydroguaiaretic acid) (catalog no. IN0590, Solarbio Life Science, China) (Yue et al., 2023). The fruits were vacuum-injected with as a control. All treated fruits were incubated under a 16-h-light -h-dark cycle at . Flesh samples from 30 fruits per treatment were collected at 14 day after infiltration and divided into three sets, each containing 10 fruits; each set was considered to be one biological replicate. The fruit flesh of each set was collected and frozen in liquid nitrogen, and stored at for experiments. 真空注射 或 ABA（目录号 862169，Sigma-Aldrich，美国）。对于 ABA 抑制剂处理，果实真空注射 NDGA（去氢愈创木脂酸）（目录号：IN0590，Solarbio Life Science，中国）（Yue et al.）果实真空注射 作为对照。所有处理过的果实均在 16 小时光照 -h -dark 循环条件下进行培养，培养箱温度为 。每个处理的 30 个果实的果肉样本在浸泡 14 天后收集，并分成三组，每组 10 个果实；每组被视为一个生物重复。收集每组果实的果肉并用液氮冷冻，然后储存在 供实验使用。
Carotenoid measurement 类胡萝卜素测量
Carotenoid was extracted from fruits, leaves or calli with acetone:hexane:ethanol (1:1:1, v/v/v). Total carotenoid was quantified using a UV-vis spectrophotometer. Carotenoids were measured and analyzed by MetWare Company (http://www.metware.cn/), and the extracts of carotenoids were measured and determined by an LC-MS/MS system (Jia et al., 2019; Dang et al., 2021). Three biological replicates were executed. A standard curve was used to quantify carotenoid levels via changing the pertinent peak area to concentration. The authentic standards of carotenoids included phytoene (CAS no. 13920-14-4, BOC, USA), phytofluene (CAS no. 27664-65-9, 13920-14-4, BOC, USA), -carotene (CAS no. 7488-99-5, BOC, USA), -cryptoxanthin (catalog no. ZES-0317S, ExtraSynthese, France), violaxanthin (catalog no. 52444, Sigma, USA), neoxanthin (CAS no. 14660-91-4, BOC, USA), -carotene (catalog no. RO19713, RHAWN, China), zeaxanthin (catalog no. BD2134, Bide, China), and lutein (catalog no. X109574, Aladdin, China). 用丙酮：正己烷：乙醇（1:1:1, v/v/v）从果实、叶片或胼胝体中提取类胡萝卜素。类胡萝卜素总量用紫外-可见分光光度计定量。类胡萝卜素由 MetWare 公司 ( http://www.metware.cn/) 进行测量和分析，类胡萝卜素提取物由 LC-MS/MS 系统进行测量和测定（Jia 等人，2019 年；Dang 等人，2021 年）。共进行了三次生物重复。通过改变相关峰面积与浓度的关系，使用标准曲线对类胡萝卜素水平进行定量。类胡萝卜素的正品标准品包括植物烯（CAS 编号：13920-14-4，美国 BOC）、植物芴（CAS 编号：27664-65-9，13920-14-4，美国 BOC）、 -胡萝卜素（CAS 编号：7488-99-5，美国 BOC）、 -隐黄素（目录编号：ZES-0317S，Extrax，美国）。ZES-0317S，ExtraSynthese，法国）、violaxanthin（目录编号：52444，Sigma，美国）、neoxanthin（CAS 编号：14660-91-4，BOC，美国）、 -胡萝卜素（目录编号：RO19713，RHAWN，中国）、玉米黄质（目录编号：BD2134，Bide，中国）和叶黄素（目录编号：X109574，Aladdin，中国）。
ABA was extracted from samples using a mixed solution of methanol:water:formic acid (15:4:1, ) supplemented with of the internal standard -ABA (catalog no. HY-100560S, MCE, USA) for quantitation. The extracts were evaporated to dryness and then dissolved in a mixed solution of methanol:water . Three biological replicates of each set of samples were executed. The extracts of ABA were measured and determined by the LC-MS/MS system (Niu et al., 2014; Liao et al., 2018). A standard curve was used to quantify carotenoid levels via changing the pertinent peak area to concentrations. The ABA standard (catalog no. 862169) was purchased from Sigma-Aldrich. 用甲醇:水:甲酸（15:4:1, ）的混合溶液提取样品中的 ABA，并加入 内标 -ABA（目录号 HY-100560S，MCE，美国）进行定量。将提取物蒸发至干，然后溶解在甲醇：水的混合溶液中 。每组样品进行三次生物重复。提取物中的 ABA 由 LC-MS/MS 系统测定（Niu 等，2014；Liao 等，2018）。通过改变相关峰面积的浓度，使用标准曲线来量化类胡萝卜素的水平。ABA 标准品（目录编号：862169）购自 Sigma-Aldrich。
RNA-seq analysis RNA-seq 分析
Total RNA was extracted from flesh of Yanfu 3 and Beni Shogun fruit at three developmental stages, 60,30 , and 0 DBFR by means of an RNAprep Pure Plant Plus Kit (catalog no. DP441, Tiangen, China) (Jia et al., 2018). Three biological replicates of each set of samples were executed, and 18 RNA-seq libraries were generated and sequenced. RNA-seq was carried out by Novogene Co. (Beijing, China). The Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA) was used for library sequencing, and RNA-seq analysis was executed as described by Jia et al. (2021). Clean reads were mapped to the apple genome sequence (https://www.rosaceae.org/malus_x_domestica/genome_GDDH13_v1.1) (Daccord et al., 2017). The RNA-seq data were submitted to GenBank of NCBI (PRJNA985276). 采用 RNAprep Pure Plant Plus Kit（产品目录号：DP441，天根公司，中国）（Jia et al.）对每组样品进行了三次生物重复，生成并测序了 18 个 RNA-seq 文库。RNA-seq由Novogene公司（中国北京）完成。文库测序采用 Illumina HiSeq 2000 平台（Illumina, San Diego, CA, USA），RNA-seq 分析按照 Jia 等人（2021 年）的描述进行。干净的读数被映射到苹果基因组序列（ https://www.rosaceae.org/malus_x_domestica/genome_GDDH13_v1.1 ）（Daccord 等人，2017 年）。RNA-seq 数据已提交至 NCBI 的 GenBank（PRJNA985276）。
RT-qPCR assay RT-qPCR 检测
25
Total RNA was extracted and RT-qPCR was executed as described in previous study (Dang et al., 2021; Jia et al., 2021). First-strand cDNA was synthesized by a PrimeScript RT reagent kit, and qPCR assay was executed using SYBR Premix Ex Taq (Takara, Japan). The relative expression levels were analyzed and determined by the method. MdEF1 MD04G1011000) and MdActin7 (MD01G1001600) were used as internal control genes (Jia et al., 2021; Ampomah-Dwamena et al., 2022). Three biological replicates were executed. The RT-qPCR primers were designed online (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are shown in Supplemental Table S3. 提取总 RNA 并按照之前的研究（Dang 等人，2021 年；Jia 等人，2021 年）进行 RT-qPCR 分析。使用 PrimeScript RT 试剂盒合成第一链 cDNA，并使用 SYBR Premix Ex Taq（日本 Takara 公司）进行 qPCR 检测。用 方法分析并确定相对表达水平。MdEF1 MD04G1011000）和 MdActin7（MD01G1001600）作为内部对照基因（Jia 等，2021；Ampomah-Dwamena 等，2022）。共进行了三次生物重复。RT-qPCR 引物是在网上 ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/) 设计的，见补充表 S3。
Cloning of gene sequences and phylogenetic analysis 基因序列的克隆和系统发育分析
The full-length coding sequence (CDS) of MdMYBS1, MdbABI5, MdLCYb or MdNCED1 were cloned from the cDNA of 'Beni Shogun' fruit flesh. The promoter sequences (listed in Supplemental Table S2) of genes including MdMYBS1, MdABI5, MdPSY2-1, MdLCYb and MdNCED1 were cloned from the DNA of young 'Beni Shogun' leaves that were collected in late spring. The prediction of cis-acting regulatory elements in gene promoters were conducted using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cis-elements of TFs were predicted using JASPAR 2020 (https://jaspar.genereg.net//) (Fornes et al., 2019; Liu et al., 2022). Protein sequences of R1-type MYBs (listed in Supplemental Table S4) deposited in GenBank of NCBI were used to construct the phylogenetic tree using MEGA version 6.0 software (Jia et al., 2021). The primers are shown in Supplemental Table S3. 从'贝尼将军'果肉的 cDNA 中克隆了 MdMYBS1、MdbABI5、MdLCYb 或 MdNCED1 的全长编码序列（CDS）。MdMYBS1、MdABI5、MdPSY2-1、MdLCYb 和 MdNCED1 等基因的启动子序列（见补充表 S2）是从春末采集的'Beni Shogun'嫩叶 DNA 中克隆的。利用 PlantCARE 数据库（http://bioinformatics.psb.ugent.be/webtools/plantcare/html/）对基因启动子中的顺式调控元件进行了预测。TFs 的顺式元件使用 JASPAR 2020 ( https://jaspar.genereg.net//) 进行预测（Forne 等人，2019；Liu 等人，2022）。使用 MEGA 6.0 版软件（Jia et al.引物见补充表 S3。
Vector construction and stable transformation of apple and tomato 苹果和番茄的载体构建和稳定转化
The CDS of MdMYBS1, MdbABI5, MdLCYb or MdNCED1 was cloned into the previously generated 26 将 MdMYBS1、MdbABI5、MdLCYb 或 MdNCED1 的 CDS 克隆到先前生成的 26
overexpression vector PRI101-FLAG (Dang et al., 2021). The RNAi constructs were obtained by cloning a 435-bp CDS fragment (between +475 and +909 bp) of MdMYBS1, a 398-bp CDS fragment (between +301 and +698 bp) of MdABI5, and a 429-bp CDS fragment (between +4 and +432 bp) of MdNCED1 into the RNAi vectors PRI101-AN and pTRV2 (Patade et al., 2014; Yue et al., 2020). Agrobacterium tumefaciens strain EHA105 harboring these constructs were transformed into apple Orin calli, apple GL-3 leaves and 'Micro-Tom' tomato plants (Jia et al., 2019; Jia et al., 2021; Zhu et al., 2023). The primers are shown in Supplemental Table S3. 过表达载体 PRI101-FLAG（Dang 等人，2021 年）。RNAi 构建物是通过克隆 MdMYBS1 的 435-bp CDS 片段（+475 和 +909 bp 之间）、MdABI5 的 398-bp CDS 片段（+301 和 +698 bp 之间）以及 MdNCED1 的 429-bp CDS 片段（+4 和 +432 bp 之间）到 RNAi 载体 PRI101-AN 和 pTRV2 中获得的（Patade 等人，2014；Yue 等人，2020）。将携带这些构建体的农杆菌菌株 EHA105 转化到苹果奥林胼胝体、苹果 GL-3 叶片和'Micro-Tom'番茄植株中（Jia 等人，2019 年；Jia 等人，2021 年；Zhu 等人，2023 年）。引物见补充表 S3。
Transient transformation of apple fruit 苹果果实的瞬时转化
Agrobacterium tumefaciens strain GV3101 cells harboring the overexpression constructs or interference constructs of MdMYBS1 and MdbABI5 were collected by centrifugation and resuspended in mixed infiltration buffer ( 5.6) containing MES, , and 200 acetosyringone. Fruits of green apple 'Granny Smith' were harvested at 140 DAFB and used for transient transformation. These Agrobacterium cultures were injected into the space between the fruit skin and flesh (An et al., 2021; Dang et al., 2021). All treated fruits were incubated at and were collected after infiltration. The infiltrated areas of the fruits were separately divided into three groups, frozen in liquid nitrogen, and stored at to analyze carotenoid content, plant hormone content, and gene expression. 农杆菌菌株 GV3101 中含有过表达构建体或 MdMYBS1 和 MdbABI5 干扰构建体的细胞经离心收集后，重悬于含有 MES、 和 200 乙酰丁香酮的混合渗透缓冲液（ 5.6）中。在 140 DAFB 时收获青苹果'Granny Smith'的果实，并用于瞬时转化。将这些农杆菌培养物注入果皮和果肉之间的空隙中（An 等人，2021 年；Dang 等人，2021 年）。所有处理过的水果都在 条件下培养，并在注入后收集 。将浸润区域的果实分成三组，用液氮冷冻，并保存在 ，以分析类胡萝卜素含量、植物激素含量和基因表达。
Subcellular localization 亚细胞定位
The fusion construct 35S:MdMYBS1-GFP was gained by cloning the CDS of MdMYBS1 (without stop codon) into the pRI101-GFP vector. Agrobacterium strain GV3101 cells containing the 27 通过将 MdMYBS1 的 CDS（不含终止密码子）克隆到 pRI101-GFP 载体中，获得了融合构建体 35S:MdMYBS1-GFP。农杆菌菌株 GV3101 细胞含有 27
35S:MdMYBS1-GFP construct were infiltrated into . benthamiana leaves and incubated for at . The GFP fluorescent signals were excited at and detected at with approximately laser value and the around 700 master gain value under a laser scanning confocal microscope (Leica, Wetzlar, Germany). DAPI (4',6-Diamidino-2-phenylindole; Invitrogen, USA) was used to stain the nucleus (Dang et al., 2021; Jia et al., 2021). 将 35S:MdMYBS1-GFP构建体渗入 . benthamiana 的叶片中，并在 ，培养 。在 处激发 GFP 荧光信号，并在 处以约 的激光值和约 700 的主增益值在激光扫描共聚焦显微镜（Leica, Wetzlar, Germany）上进行检测。DAPI（4',6-二氨基-2-苯基吲哚；Invitrogen，美国）用于染色细胞核（Dang 等，2021；Jia 等，2021）。
GUS activity analysis GUS 活性分析
The Pro:GUS fusion vector ProMdMYBS1:GUS or ProMdABI5:GUS was generated by cloning the promoter fragment of MdMYBS1 (2016 bp) or MdABI5 (2005 bp) into pCAMBIA1301 (primers are shown in Supplemental Table S3). Agrobacterium GV3101 cells harboring the Pro:GUS fusion constructs were infiltrated into apple calli and N. benthamiana leaves (Jia et al., 2021; Zhu et al., 2023). Three biological replicates were executed. GUS staining was conducted using a -Galactosidase Reporter Gene Staining Kit (Solarbio, China). 将 MdMYBS1（2016 bp）或 MdABI5（2005 bp）的启动子片段克隆到 pCAMBIA1301 中，产生 Pro:GUS 融合载体 ProMdMYBS1:GUS 或 ProMdABI5:GUS（引物见补充表 S3）。将携带 Pro:GUS 融合构建体的农杆菌 GV3101 细胞渗入苹果胼胝体和 N. benthamiana 叶片中（Jia 等人，2021 年；Zhu 等人，2023 年）。共进行了三次生物重复。使用 -Galactosidase 报告基因染色试剂盒（Solarbio，中国）进行 GUS 染色。
Dual-luciferase (LUC) assay 双荧光素酶（LUC）测定
A series of promoter-LUC reporter constructs was obtained by cloning the promoter fragment of MdABI5, MdMYBS1, MdPSY2-1, MdLCYb, or MdNCED1 into pGreenll 0800-LUC vector. The effector construct 35S:MdMYBS1 or 35S:MdABI5 was obtained by cloning the CDS of MdMYBS1 or MdABI5 into the effector vector pGreenll 62-SK (primers are shown in Supplemental Table S3). The effectors with their corresponding reporters were co-infiltrated into the . benthamiana leaves. The luciferase activities of firefly and Renilla (LUC/REN ratios) were analyzed at after the infiltration using the Dual-Luciferase Reporter Assay System (Promega, USA) (Dang et al., 28 通过将 MdABI5、MdMYBS1、MdPSY2-1、MdLCYb 或 MdNCED1 的启动子片段克隆到 pGreenll 0800-LUC 载体中，获得一系列启动子-LUC 报告构建体。通过将 MdMYBS1 或 MdABI5 的 CDS 克隆到效应子载体 pGreenll 62-SK 中，得到效应子构建体 35S:MdMYBS1 或 35S:MdABI5 （引物如补充表 S3 所示）。将效应子及其相应的报告子共同渗入 . benthamiana 的叶片中。渗入后，使用双荧光素酶报告分析系统（Dual-Luciferase Reporter Assay System，Promega，USA）在 分析萤火虫和 Renilla 的荧光素酶活性（LUC/REN 比率）（Dang 等，28）。
2021; Jia et al., 2021; Zhu et al., 2023). Six biological replicates were executed. For ABA treatment, the infiltrated leaves were sprayed with before analyzing. 2021；Jia 等人，2021；Zhu 等人，2023）。共进行了 6 次生物重复。对于 ABA 处理，在分析前用 喷洒浸润的叶片。
Y1H screening of the cDNA library cDNA 文库的 Y1H 筛选
Y1H screening was performed using a previously constructed apple fruit cDNA library (Jia et al., 2021). The BD-MdMYBS1 construct was obtained by cloning a 473-bp promoter fragment (between -2016 and -1544 bp) of MdMYBS1 into the reporter vector pAbAi (primers are shown in Supplemental Table S3) and introduced into Y1H-susceptible yeast. Next, the AD-cDNA library was introduced into susceptible yeast with BD-MdMYBS1 to screen and identify the specific binding of TFs. 利用之前构建的苹果果实 cDNA 文库（Jia 等人，2021 年）进行了 Y1H 筛选。BD-MdMYBS1构建体是通过将MdMYBS1的473 bp启动子片段（-2016和-1544 bp之间）克隆到报告载体pAbAi中获得的（引物如补充表S3所示），并导入Y1H敏感酵母中。接下来，将 AD-cDNA 文库与 BD-MdMYBS1 一起导入易感酵母，以筛选和鉴定 TF 的特异性结合。
For the Y1H assay (Dang et al., 2021; Jia et al., 2021), the CDS of MdMYBS1 or MdABI5 was cloned into the effector vector pJG4-5 or pGADT7 (Clontech, USA). The promoter fragment of MdPSY2-1, MdLCYb, or MdNCED1 was cloned into the reporter vector pLacZi (Clontech, USA), and the MdMYBS1 promoter fragment was cloned into the reporter vector pAbAi (Clontech, USA). The primers are listed in Supplemental Table S3. 为了进行 Y1H 试验（Dang 等，2021；Jia 等，2021），将 MdMYBS1 或 MdABI5 的 CDS 克隆到效应载体 pJG4-5 或 pGADT7（美国 Clontech 公司）中。将 MdPSY2-1、MdLCYb 或 MdNCED1 的启动子片段克隆到报告载体 pLacZi（美国 Clontech 公司）中，将 MdMYBS1 启动子片段克隆到报告载体 pAbAi（美国 Clontech 公司）中。引物见补充表 S3。
For the EMSA, the fusion construct MdMYBS1-GST or MdABI5-GST with GST-tag were obtained by cloning the CDS of MdMYBS1 or MdABI5 into pGEX-4T-1 vector, and then separately introduced into Escherichia coli cells BL21 (DE3) for protein production. The and Biotin end-labeled DNA probes for EMSA were synthesized by the Sangon Biotech (China). The EMSA 在 EMSA 中，将 MdMYBS1 或 MdABI5 的 CDS 克隆到 pGEX-4T-1 载体中，得到带有 GST 标记的 MdMYBS1-GST 或 MdABI5-GST 融合构建体，然后分别导入大肠杆菌 BL21 (DE3) 细胞中生产蛋白质。用于 EMSA 的 和 生物素末端标记 DNA 探针由中国生工生物技术有限公司合成。EMSA
was carried out by a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, USA) as described in previous study (Dang et al., 2021; Jia et al., 2021). The primers and probe sequences for EMSA are shown in Supplemental Table S3. 按照之前研究（Dang 等，2021；Jia 等，2021）的描述，使用 LightShift 化学发光 EMSA 试剂盒（Thermo Fisher Scientific, USA）进行了检测。EMSA 的引物和探针序列见补充表 S3。
For the ChIP-PCR analysis, the fusion construct 35S:MdMYBS1-GFP or 35S:MdABI5-GFP with GFP tag were obtained by cloning the CDS of MdMYBS1 or MdABI5 into the pRI101-GFP vector. GFP-tag transgenic calli were used for the ChIP-PCR assay (Dang et al., 2021; Jia et al., 2021; Zhu et al., 2023), which was carried out using an EpiQuik Plant ChIP Kit (EpiGentek, USA). The chromatin DNA fragments were incubated with anti-GFP antibody (Abcam, UK), and then qPCR was used to examine the enrichment of immunoprecipitated chromatin. MdACTIN served as the internal control to normalize the ChIP enrichment. Three biological replicates were executed. Primers used for ChIP-PCR are shown in Supplemental Table S3. 为了进行 ChIP-PCR 分析，将 MdMYBS1 或 MdABI5 的 CDS 克隆到 pRI101-GFP 载体中，得到带有 GFP 标记的融合构建体 35S:MdMYBS1-GFP 或 35S:MdABI5-GFP。GFP 标记的转基因胼胝体用于 ChIP-PCR 检测（Dang 等，2021；Jia 等，2021；Zhu 等，2023），该检测使用 EpiQuik Plant ChIP Kit（EpiGentek，美国）进行。染色质 DNA 片段与抗-GFP 抗体（Abcam，英国）孵育，然后使用 qPCR 检测免疫沉淀染色质的富集。MdACTIN 作为内部对照，对 ChIP 富集进行归一化处理。共进行了三次生物重复。用于 ChIP-PCR 的引物见补充表 S3。
Statistical analyses 统计分析
The data from the experiments in this study were obtained using three independent biological replicates. Significant differences between samples were analyzed by Student's -test , ), and differences among samples were analyzed by one-way analysis of variance (ANOVA) using GraphPad 9.0 software and Microsoft Excel 2019. 本研究的实验数据是通过三个独立的生物重复获得的。样本间的显著差异通过学生 -test , ) 进行分析，样本间的差异通过单因素方差分析（ANOVA） ，使用 GraphPad 9.0 软件和 Microsoft Excel 2019。
Accession numbers 编号
The accessions of the sequence of the genes and proteins used in this study are deposited in Malus domestica genome (GDDH13 v1.1) database and GenBank database of NCBI: MdMYBS1 本研究中使用的基因和蛋白质的序列登录在 Malus domestica 基因组（GDDH13 v1.1）数据库和 NCBI 的 GenBank 数据库中：MdMYBS1
(MD05G1282700), MdABI5 (MD14G1021600). Protein sequences of R1-type MYBs are listed in Supplemental Table . (MD05G1282700）、MdABI5（MD14G1021600）。R1 型 MYB 的蛋白质序列列于补充表 中。
Supplemental Data 补充数据
Supplemental Figure S1. The concentrations of total flavonoid and total carotenoid. 补充图 S1.总黄酮和总类胡萝卜素的浓度。
Supplemental Figure S2. The mass spectrum of carotenoid compounds and abscisic acid (ABA) using LC-MS/MS. 补充图 S2。使用 LC-MS/MS 分析类胡萝卜素化合物和脱落酸（ABA）的质谱。
Supplemental Figure S3. Levels of carotenoid compounds identified by LC-MS/MS of 'Yanfu 3' and 'Beni Shogun' fruits at 60, 30, and 0 days before fruit ripening (DBFR). 补充图 S3。在果实成熟前 60 天、30 天和 0 天（DBFR），通过 LC-MS/MS 鉴定的'盐富 3 号'和'贝尼将军'果实中类胡萝卜素化合物的含量。
Supplemental Figure S4. RNA-seq analysis for fruit flesh of 'Yanfu 3' and 'Beni Shogun' at three ripening stages of 60,30 and 0 days before fruit ripening (DBFR). 补充图 S4.对'盐富 3 号'和'贝尼将军'在果实成熟前 60 天、30 天和 0 天（DBFR）三个成熟阶段果肉的 RNA-seq 分析。
Supplemental Figure S5. The screening of candidate MYB-related transcription factors. 补充图 S5.候选 MYB 相关转录因子的筛选。
Supplemental Figure S6. The expression pattern of MdMYBS1 in apple leaf, flower and fruit of 'Yanfu 3' and 'Beni Shogun'. 补充图 S6.MdMYBS1 在'盐富 3 号'和'贝尼将军'苹果叶片、花和果实中的表达模式。
Supplemental Figure S7. The expression pattern of MdMYBS1 in ripened fruit flesh of 13 Malus genotypes. 补充图 S7。13 个马蔺基因型成熟果肉中 MdMYBS1 的表达模式。
Supplemental Figure S8. The screening of carotenoid and abscisic acid (ABA) biosynthetic genes. 31 补充图 S8。类胡萝卜素和脱落酸（ABA）生物合成基因的筛选。31
Supplemental Figure S10. Overexpressing MdLCYb in apple calli influences carotenoid biosynthesis. 补充图 S10.在苹果胼胝体中过表达 MdLCYb 会影响类胡萝卜素的生物合成。
Supplemental Figure S11. The Y1H assay was performed to show that MdMYBS1 binds to the promoters of MdPSY2-1, MdLCYb and MdNCED1. 补充图 S11。Y1H 试验表明，MdMYBS1 与 MdPSY2-1、MdLCYb 和 MdNCED1 的启动子结合。
Supplemental Figure S12. The identification of candidate MdABI5 transcription factor. 补充图 S12.候选 MdABI5 转录因子的鉴定。
Supplemental Table S1. Pearson's correlation between MYB TFs transcript levels and content of total carotenoid, phytoene, -Carotene, -Cryptoxanthin or ABA in 'Yanfu 3' and 'Beni Shogun' fruit flesh. 补充表 S1.'盐富 3 号'和'贝尼将军'果肉中 MYB TFs 转录水平与总类胡萝卜素、植物色素、 -胡萝卜素、 -隐黄素或 ABA 含量之间的皮尔逊相关性。
Supplemental Table S2. Promoter sequences analyzed in the study. 补充表 S2。研究中分析的启动子序列。
Supplemental Table S3. Primer and probe sequences used in the study. 补充表 S3。研究中使用的引物和探针序列。
Supplemental Table S4. Protein sequences of R1-type MYB TFs were used to construct the phylogenetic tree. 补充表 S4.用于构建系统发生树的 R1 型 MYB TF 蛋白序列。
Funding information 资金信息
This work was supported by the National Natural Science Foundation of China (31972362, U22A20493, 32272657, 32001993), the Project of the Shandong Natural Science Foundation (ZR2020QC143), the Taishan Scholar Foundation of Shandong Province (tstp20221134), and the 32 本研究得到了国家自然科学基金项目（31972362、U22A20493、32272657、32001993）、山东省自然科学基金项目（ZR2020QC143）、山东省泰山学者基金项目（tstp20221134）和山东省自然科学基金项目（ZR2020QC143）的资助。
Shandong Key R&D Plan (Agricultural Variety Project) (2022LZGCQY008). 山东省重点研发计划（农业品种项目）（2022LZGCQY008）。
Acknowledgments 致谢
We thank the Laixi breeding farm and Laixi Gaosong farm (Qingdao City, China) for providing experimental resources. We thank Planteditors (www.planteditors.com) for editing this manuscript. 感谢莱西育种场和莱西高松农场（中国青岛市）提供的实验资源。感谢 Planteditors ( www.planteditors.com) 对本稿的编辑。
Author contributions 作者供稿
D.J., and Y.Y. designed the experiments and analysis; D.J., Y.L., K.J., B.H., Q.D., H.W., X.W., and C.L. performed the experiments; D.J., Y.Z., and J.N. carried out data analyses; Y.Y., Y.Z., and J.N. contributed materials/analysis tools; D.J., and Y.Y. wrote the article. All authors read and approved the manuscript. D.J.和Y.Y.设计了实验和分析；D.J.、Y.L.、K.J.、B.H.、Q.D.、H.W.、X.W.和C.L.进行了实验；D.J.、Y.Z.和J.N.进行了数据分析；Y.Y.、Y.Z.和J.N.提供了材料/分析工具；D.J.和Y.Y.撰写了文章。所有作者阅读并批准了手稿。
Conflicts of interest 利益冲突
The authors declare no conflict of interest. 作者声明没有利益冲突。
Data availability statement 数据可用性声明
The data that support the findings of this study are available from the corresponding author upon request. 支持本研究结果的数据可向通讯作者索取。
33
Figure legends 图例
Figure 1 Identification of carotenoid compounds and carotenoid-related regulators in 'Yanfu 3' and 'Beni Shogun' fruits. 图 1 "盐富 3 号 "和 "贝尼将军 "果实中类胡萝卜素化合物和类胡萝卜素相关调节因子的鉴定。
A, Phenotypes of 'Yanfu 3' and 'Beni Shogun' fruits harvested at 60, 30, and 0 days before fruit ripening (DBFR). Scale bars, . Fruit images were digitally extracted for comparison. B-H, Levels of the carotenoid compounds phytoene (B), -carotene (C), -cryptoxanthin (D), violaxanthin (E), neoxanthin (F) and lutein (G), and of abscisic acid (ABA) (H) were measured using LC-MS/MS. I-L, Relative expression levels of MdMYBS1 (I), MdPSY2-1 (J), MdLCYb (K), and MdNCED1 (L) were determined using RT-qPCR. At each harvesting time, 18 fruits were divided into three sets, each containing six fruits, and each set was considered to be one biological replicate. The data bars represent mean standard deviation. The error bars represent standard deviations of 3 independent biological replicates, and six fruits measured for each replicate. Asterisks indicate significant differences analyzed by Student's -test ; ns, not significant. A, 在果实成熟前 60 天、30 天和 0 天（DBFR）采收的'烟富 3 号'和'贝尼将军'果实的表型。比例尺， 。对果实图像进行了数字提取以进行比较。B-H，类胡萝卜素化合物 phytoene (B)、 -胡萝卜素 (C)、 -隐黄素 (D)、violaxanthin (E)、neoxanthin (F) 和叶黄素 (G) 的含量，以及脱落酸 (ABA) (H) 的含量是通过 LC-MS/MS 测定的。I-L，使用 RT-qPCR 测定 MdMYBS1 (I)、MdPSY2-1 (J)、MdLCYb (K) 和 MdNCED1 (L) 的相对表达水平。在每个采收期，18 个果实被分成三组，每组包含 6 个果实，每组被视为一个生物重复。数据条代表平均值 标准偏差。误差条代表 3 个独立生物重复的标准偏差，每个重复测量 6 个果实。星号表示经学生 -test 分析的显著差异；ns 表示不显著。
Figure 2 Characterization and subcellular localization of MdMYBS1. 图 2 MdMYBS1 的特征和亚细胞定位。
A, MdMYBS1 contains a central SANT/MYB DNA-binding domain and an ZnF_C2HC domain, determined on the SMART website (http://smart.embl-heidelberg.de/). B, Phylogenetic analysis of MdMYBS1 indicated that the amino acid sequence of MdMYBS1 was most similar to that of an Arabidopsis protein KUA1. The horizontal branch length represents the proportional to the estimated number of amino acids substitutions per residue ( aa substitution per residue). 34 A, MdMYBS1 包含一个中央 SANT/MYB DNA 结合结构域和一个 ZnF_C2HC 结构域，由 SMART 网站 ( http://smart.embl-heidelberg.de/) 测定。B，MdMYBS1 的系统进化分析表明，MdMYBS1 的氨基酸序列与拟南芥蛋白 KUA1 的氨基酸序列最为相似。水平分支长度与估计的每个残基的氨基酸取代数成正比（ aa/ residue）。34
Protein sequences of R1-type MYBs deposited in GenBank of NCBI were used to construct the phylogenetic tree are listed in Table S4. C, The DNA binding region of MdMYBS1 includes a conserved SHAQKYF motif of R1-type MYB TFs like AtKUA1, OsMYBS3 and OsMYBS2. AtKUA1 and OsMYBS3 contain an EAR motif (LxLxL), and MdMYBS1 and OsMYBS2 without the EAR motif. D, The MdMYBS1-GFP fusion protein derived from the 35S promoter (Pro35S:MdMYBS1-GFP) and the control 35S:GFP were transiently expressed in Nicotiana benthamiana leaves. 6-Diamidino-2-phenylindole (DAPI) was used to stain the nucleus. The fluorescent images were observed and obtained using confocal microscopy. Scale bars um. 表 S4 列出了保存在 NCBI GenBank 中的 R1 型 MYB 蛋白序列，用于构建系统发生树。C，MdMYBS1 的 DNA 结合区包括 R1 型 MYB TFs（如 AtKUA1、OsMYBS3 和 OsMYBS2）保守的 SHAQKYF 基序。AtKUA1 和 OsMYBS3 含有一个 EAR 基序（LxLxL），而 MdMYBS1 和 OsMYBS2 没有 EAR 基序。D，瞬时表达来自 35S 启动子的 MdMYBS1-GFP 融合蛋白（Pro35S:MdMYBS1-GFP）和对照 35S:GFP。用 6-二脒基-2-苯基吲哚（DAPI）对细胞核染色。使用共聚焦显微镜观察并获得荧光图像。标尺条 um。
Figure 3 MdMYBS1 promotes carotenoid biosynthesis and endogenous abscisic acid (ABA) accumulation in transient transgenic apple fruits, transgenic apple calli and plants, and tomato fruits. 图 3 MdMYBS1 在瞬时转基因苹果果实、转基因苹果胼胝体和植株以及番茄果实中促进类胡萝卜素的生物合成和内源脱落酸 (ABA) 的积累。
A, T-DNA region of the vectors MdMYBS1-OVX, MdMYBS1-RNAi and MdMYBS1-TRV2 used for transformation. B, MdMYBS1 was overexpressed (MdMYBS1-OVX) and silenced (MdMYBS1-TRV) in transient transgenic 'Granny Smith' apple fruits. P101F or TRV transient transgenic apple fruits transformed with empty vector PRI101-flag or pTRV2. Scale bars, . Fruit images were digitally extracted for comparison. C-F, Levels of MdMYBS1 expression, total carotenoid, phytoene, -carotene, -cryptoxanthin, violaxanthin, neoxanthin and ABA in transient transgenic apple fruits. G, MdMYBS1 was overexpressed and silenced (MdMYBS1-RNAi) in apple calli. P101F or P101R transgenic apple calli transformed with empty vector PRI101-flag or PRI101-AN. Scale bars, , Levels of MdMYBS1 expression, total carotenoid, phytoene, -carotene, -cryptoxanthin, violaxanthin, neoxanthin and ABA in transgenic apple calli. L, MdMYBS1 was 35 A. 用于转化的载体 MdMYBS1-OVX、MdMYBS1-RNAi 和 MdMYBS1-TRV2 的 T-DNA 区域。B，在转基因 "Granny Smith "苹果果实中过表达（MdMYBS1-OVX）和沉默（MdMYBS1-TRV）MdMYBS1。用空载体 PRI101-flag 或 pTRV2 转化的 P101F 或 TRV 转基因苹果果实。比例尺， 。果实图像通过数字提取进行比较。C-F，转基因苹果果实中 MdMYBS1 的表达、类胡萝卜素总量、植物色素、 -胡萝卜素、 -隐黄素、violaxanthin、neoxanthin 和 ABA 的水平。G，在苹果胼胝体中过表达和沉默（MdMYBS1-RNAi）MdMYBS1。用空载体 PRI101-flag 或 PRI101-AN 转化的 P101F 或 P101R 转基因苹果胼胝体。标尺条， ，转基因苹果胼胝体中 MdMYBS1 表达、类胡萝卜素总量、植物烯、 -胡萝卜素、 -隐黄素、小叶黄素、新黄素和 ABA 的水平。L, MdMYBS1 为 35
overexpressed in apple plants. M-P, Levels of MdMYBS1 expression, total carotenoid, phytoene, -carotene, -cryptoxanthin, neoxanthin and ABA in transgenic apple leaves. was heterologously overexpressed in tomato fruits. Scale bars, , Levels of MdMYBS1 expression, total carotenoid, phytoene, lycopene, -carotene and ABA in transgenic tomato fruits. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test ; ns, not significant. 在苹果植株中过表达。M-P，转基因苹果叶片中 MdMYBS1 的表达、类胡萝卜素总量、植酸、 -胡萝卜素、 -隐黄素、新黄素和 ABA 的水平。 在番茄果实中异源过表达。标尺条， ，转基因番茄果实中 MdMYBS1 的表达水平、类胡萝卜素总量、植物红素、番茄红素、 -胡萝卜素和 ABA。数据条代表平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。星号表示经学生 -test 分析的显著差异；ns 表示不显著。
Figure 4 MdMYBS1 promotes the transcription of the carotenoid biosynthetic genes MdPSY2-1 and MdLCYb. 图 4 MdMYBS1 促进类胡萝卜素生物合成基因 MdPSY2-1 和 MdLCYb 的转录。
A, Expression levels of MdPSY2-1 and MdLCYb in MdMYBS1-overexpressing (MdMYBS1-OVX) or MdMYBS1-silenced (MdMYBS1-RNAi or MdMYBS1-TRV) apple fruits, calli and leaves. P101F, P101R or TRV transgenic lines transformed with empty vector PRI101-flag, PRI101-AN or pTRV2. B, ChIP-PCR showing that MdMYBS1 binds to the promoters of MdPSY2-1 and MdLCYb containing the major motif TTATC(YD) or (HY)TATCCA in vivo. Five regions (SO-S4) in MdPSY2-1, and five regions (SO-S4) in MdLCYb were investigated. C, EMSA of MdMYBS1 direct binding to the TTTATCTA motif of the MdPSY2-1 promoter, and the CTTATCCA motif of the MdLCYb promoter. D, Dual-luciferase assay showing that MdMYBS1 positively regulates MdPSY2-1 and MdLCYb promoter activity. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test ( ); ns, not significant. A, MdPSY2-1 和 MdLCYb 在 MdMYBS1-overexpressing (MdMYBS1-OVX) 或 MdMYBS1-silenced (MdMYBS1-RNAi 或 MdMYBS1-TRV) 苹果果实、胼胝体和叶片中的表达水平。用空载体 PRI101-flag、PRI101-AN 或 pTRV2 转化的 P101F、P101R 或 TRV 转基因品系。B，ChIP-PCR 显示 MdMYBS1 与 MdPSY2-1 和 MdLCYb 的启动子结合，这些启动子含有 TTATC(YD) 或 (HY)TATCCA 主基调。研究了 MdPSY2-1 的五个区域（SO-S4）和 MdLCYb 的五个区域（SO-S4）。C、MdMYBS1 与 MdPSY2-1 启动子的 TTTATCTA 基序和 MdLCYb 启动子的 CTTATCCA 基序直接结合的 EMSA。D，双荧光素酶检测显示 MdMYBS1 正向调节 MdPSY2-1 和 MdLCYb 启动子的活性。数据条代表平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。星号表示经学生 -test ( ) 分析的显著差异；ns 表示不显著。
Figure 5 MdMYBS1 promotes the transcription of the abscisic acid (ABA) biosynthetic gene MdNCED1. 图 5 MdMYBS1 促进脱落酸（ABA）生物合成基因 MdNCED1 的转录。
A, Expression levels of MdNCED1 in MdMYBS1-overexpressing (MdMYBS1-OVX) or MdMYBS1-silenced (MdMYBS1-RNAi or MdMYBS1-TRV) apple fruits, calli and leaves. P101F, P101R or TRV transgenic lines transformed with empty vector PRI101-flag, PRI101-AN or pTRV2. B, MdNCED1 was overexpressed (MdNCED1-OVX) and silenced (MdNCED1-RNAi) in apple calli. P101F or P101R transgenic apple calli transformed with empty vector PRI101-flag or PRI101-AN. C-E, Levels of MdNCED1 expression, ABA, phytoene, -carotene, -cryptoxanthin, violaxanthin and neoxanthin in transgenic apple calli. F, ChIP-PCR showing that MdMYBS1 binds to the MdNCED1 promoter containing the major motif TTATC(YD) or (HY)TATCCA in vivo. Five regions (S0-S4) in MdNCED1 were investigated. G, EMSA of MdMYBS1 direct binding to the ATTATCCT motif of the MdNCED1 promoter. H, Dual-luciferase assay showing that MdMYBS1 positively regulates the MdNCED1 promoter activity. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test ( ); ns, not significant. Figure 6 The MdMYBS1 promoter was activated by exogenous abscisic acid (ABA) and MdABI5. A, 苹果果实、胼胝体和叶中 MdMYBS1-overexpressing (MdMYBS1-OVX) 或 MdMYBS1-silenced (MdMYBS1-RNAi 或 MdMYBS1-TRV) 的 MdNCED1 表达水平。用空载体 PRI101-flag、PRI101-AN 或 pTRV2 转化的 P101F、P101R 或 TRV 转基因品系。B. 在苹果胼胝体中过表达（MdNCED1-OVX）或沉默（MdNCED1-RNAi）MdNCED1。用空载体 PRI101-flag 或 PRI101-AN 转化 P101F 或 P101R 转基因苹果胼胝体。C-E，转基因苹果胼胝体中 MdNCED1 的表达、ABA、植物烯、 -胡萝卜素、 -隐黄素、violaxanthin 和 neoxanthin 的水平。F、ChIP-PCR 显示 MdMYBS1 在体内与含有主要基序 TTATC(YD) 或 (HY)TATCCA 的 MdNCED1 启动子结合。研究了 MdNCED1 的五个区域（S0-S4）。G，MdMYBS1 与 MdNCED1 启动子的 ATTATCCT 主题直接结合的 EMSA。H，双荧光素酶检测显示 MdMYBS1 正向调节 MdNCED1 启动子的活性。数据条代表平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。星号表示经学生 -test ( ) 分析的显著差异；ns 表示不显著。图 6 外源脱落酸（ABA）和 MdABI5 激活了 MdMYBS1 启动子。
A, Dual LUC assay showing that the relative LUC/REN activity of the MdMYBS1 promoter increased significantly in Nicotiana benthamiana leaves under ABA treatment compared to the control ( treatment). B, GUS assay showing that the relative GUS activity of the MdMYBS1 promoter increased significantly in transgenic apple calli under treatment A, 双 LUC 检测表明，与对照（ ）相比，在 ABA 处理下，烟草叶片中 MdMYBS1 启动子的相对 LUC/REN 活性显著增加。B, GUS 检测表明，在 处理下，转基因苹果胼胝体中 MdMYBS1 启动子的相对 GUS 活性明显增加。
compared to the control. C, Y1H assay confirming that MdABI5 binds to the MdMYBS1 promoter. D, ChIP-PCR confirming that MdABI5 binds to the promoter of MdMYBS1 containing the major ABRE motif in vivo. Four regions (S0-S3) in MdMYBS1 promoter were investigated. E, EMSA confirming that MdABI5 directly binds to the ABRE motif TACGTG (S3) in the MdMYBS1 promoter. F and G, Dual LUC assay (F) and GUS activity assay (G) showing that MdABI5 positively regulates MdMYBS1 promoter activity. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test ( ); ns, not significant. 与对照组相比。C、Y1H 检测证实 MdABI5 与 MdMYBS1 启动子结合。D、ChIP-PCR 证实 MdABI5 与体内含有主要 ABRE motif 的 MdMYBS1 启动子结合。研究了 MdMYBS1 启动子中的四个区域（S0-S3）。E，EMSA 证实 MdABI5 直接与 MdMYBS1 启动子中的 ABRE 主题 TACGTG（S3）结合。F 和 G，双重 LUC 检测（F）和 GUS 活性检测（G）显示 MdABI5 正向调节 MdMYBS1 启动子的活性。数据条代表平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。星号表示经 Student's -test ( ) 分析的显著差异；ns 表示不显著。
Figure 7 MdABI5 promotes carotenoid and abscisic acid (ABA) accumulation and responds to ABA. 图 7 MdABI5 促进类胡萝卜素和脱落酸（ABA）的积累并对 ABA 作出反应。
A, T-DNA region of the vectors MdABI5-OVX, MdABI5-RNAi and MdABI5-TRV2 used for transformation. B, MdABI5 was overexpressed (MdABI5-OVX) and silenced (MdABI5-TRV) in transient transgenic 'Granny Smith' apple fruits. P101F or TRV transient transgenic apple fruits transformed with empty vector PRI101-flag or pTRV2. Fruit images were digitally extracted for comparison. Scale bars, and , Levels of total carotenoid and ABA in transient transgenic apple fruits. E, Expression levels of MdABI5, MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1 in transient transgenic apple fruits. F, MAABI5 was overexpressed and silenced (MdABI5-RNAi) in apple calli. P101F or P101R transgenic apple calli transformed with empty vector PRI101-flag or PRI101-AN. G and H, Levels of total carotenoid and ABA in transgenic apple calli. I, Expression levels of MdAB15, MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1 in transgenic apple calli. J, Dual LUC assay showing that the relative LUC/REN activity of the MdABI5 promoter significantly 38 A. 用于转化的载体 MdABI5-OVX、MdABI5-RNAi 和 MdABI5-TRV2 的 T-DNA 区域。B、MdABI5 在转基因'Granny Smith'苹果果实中的过表达（MdABI5-OVX）和沉默（MdABI5-TRV）。用空载体 PRI101-flag 或 pTRV2 转化的 P101F 或 TRV 转基因苹果果实。果实图像经数字提取后进行比较。比例尺， 和 ，类胡萝卜素总量和 ABA 在瞬时转基因苹果果实中的水平。E，转基因苹果果实中 MdABI5、MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1 的表达水平。F、苹果胼胝体中 MAABI5 的过表达和沉默（MdABI5-RNAi）。用空载体 PRI101-flag 或 PRI101-AN 转化的 P101F 或 P101R 转基因苹果胼胝体。G 和 H，转基因苹果胼胝体中总类胡萝卜素和 ABA 的水平。I, 转基因苹果胼胝体中 MdAB15、MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1 的表达水平。J, 双 LUC 检测显示 MdABI5 启动子的相对 LUC/REN 活性明显高于 MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1。
increased in Nicotiana benthamiana leaves under ABA treatment compared to the control. K, GUS assay showing that the relative GUS activity of the MdABI5 promoter significantly increased in transgenic apple calli under treatment compared to the control. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test . ABA 处理下的烟草叶片中的 MdABI5 启动子活性与对照组相比有所增加。K, GUS 检测显示，在 处理下，与对照相比，转基因苹果胼胝体中 MdABI5 启动子的相对 GUS 活性显著增加。数据条代表平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。星号表示经学生 -test 分析的显著差异。
A, The phenotypes of MdABI5-OVX apple calli with silencing of MdMYBS1 (MdMYBS1-RNAi) and MdMYBS1-OVX apple calli with silencing of MdABI5 (MdABI5-RNAi). Scale bars, , Transcription levels of MdABI5 and MdMYBS1 in transgenic apple calli. C and D, Total carotenoid content and ABA content in transgenic apple calli. E, Expression levels of MdPSY2-1, MdLCYb and MdNCED1 in transgenic apple calli. The data bars represent mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Asterisks indicate significant differences analyzed by Student's -test ; ns, not significant. A, 沉默 MdMYBS1 的 MdABI5-OVX 苹果胼胝体（MdMYBS1-RNAi）和沉默 MdABI5 的 MdMYBS1-OVX 苹果胼胝体（MdABI5-RNAi）的表型。标尺条， ，转基因苹果胼胝体中 MdABI5 和 MdMYBS1 的转录水平。C 和 D，转基因苹果胼胝体中类胡萝卜素总含量和 ABA 含量。E，转基因苹果胼胝体中 MdPSY2-1、MdLCYb 和 MdNCED1 的表达水平。数据条代表平均值 标准差，误差条代表 3 个独立生物重复的标准差。星号表示经学生 -test 分析的显著差异；ns 表示不显著。
Figure 9 Exogenous abscisic acid (ABA) promotes carotenoid accumulation in apple fruit. 图 9 外源脱落酸（ABA）促进苹果果实中类胡萝卜素的积累。
A, 'Orin' apple fruits were treated with different concentrations and of exogenous ABA; nordihydroguaiaretic acid (NDGA) was used as an ABA inhibitor. Fruit images were digitally extracted for comparison. Scale bars, . and , Levels of and total carotenoid content (C) in treated fruit flesh. , The relative expression levels of , MdMYBS1, MdPSY2-1, MdLCYb and MdNCED1 in treated fruit flesh. The data bars represent 39 A, 'Orin' 苹果果实用不同浓度的外源 ABA 和 处理； nordihydroguaiaretic acid (NDGA) 用作 ABA 抑制剂。对果实图像进行数字提取以进行比较。比例尺， 。 和 ，处理后果肉中 和类胡萝卜素总含量 (C) 的水平。 处理果肉中 、MdMYBS1、MdPSY2-1、MdLCYb 和 MdNCED1 的相对表达水平。数据条代表 39
mean standard deviation, and the error bars represent standard deviations of 3 independent biological replicates. Different letters represent significant differences at by a Tukey's multiple range test using one-way ANOVA. 平均值 标准偏差，误差条代表 3 个独立生物重复的标准偏差。不同字母代表 ，通过单因素方差分析的 Tukey's 多重范围检验，差异显著。
Figure 10 Proposed model for the roles of the MdABI5-MdMYBS1 cascade activated by abscisic acid in regulating carotenoid-derived fruit coloration and accumulation in apple. 图 10 脱落酸 激活的 MdABI5-MdMYBS1 级联在调控苹果类胡萝卜素衍生果实着色和 积累中的作用的拟议模型。
Carotenoid is the main factor influencing fruit flesh color. MdMYBS1 contributes to carotenoid accumulation via activating carotenogenic genes MdPSY2-1 and MdLCYb. MdMYBS1 enhances ABA accumulation by activating MdNCED1 expression. In addition, MdABI5 activates MdMYBS1 expression by binding its promoter to increase carotenoid and ABA accumulation. Furthermore, exogenous ABA promotes carotenoid biosynthesis and enhances MdMYBS1 and MdABI5 transcription by activating their promoter activities. 类胡萝卜素是影响果肉颜色的主要因素。MdMYBS1 通过激活类胡萝卜素生成基因 MdPSY2-1 和 MdLCYb 来促进类胡萝卜素的积累。MdMYBS1 通过激活 MdNCED1 的表达来增强 ABA 的积累。此外，MdABI5 通过结合 MdMYBS1 的启动子激活其表达，从而增加类胡萝卜素和 ABA 的积累。此外，外源 ABA 可促进类胡萝卜素的生物合成，并通过激活 MdMYBS1 和 MdABI5 的启动子活性来增强它们的转录。
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D
Withóout EAR motif a 没有 EAR motif a
Pro35S:MdMYBS1-GFP
Figure 3. 图 3.
B
eson 埃森
G
H
I
L
N
0
Figure 4. 图 4.
A
Hot probe 热探头
Cold probe 冷探头
Mutant hot probe 突变热探针
Mutant cold probe 变异冷探针
D
Figure 5. 图 5.
A
Transient transgenic apple fruits 转基因苹果果实
Transgenic apple leaves 转基因苹果叶
D
E
ProMdNCED1
H Pro35S MaMYBS1
Figure 6. 图 6.
A
B
C
D ATG D ATG
E
ProMdMYBS1
MdABI5-GST Hot probe Cold probe MdABI5-GST 热探头 冷探头
Mutant hot probe Mutant hot probe 突变热探针 突变热探针
Mutant cold probe 变异冷探针
F
G
Figure 7. 图 7.
F
J
Relative LUC/REN activity 相对 LUC/REN 活性
ProMAABI5
Figure 8. 图 8.
B
E
Figure 9. 图 9.
Figure 10. 图 10.
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