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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。
13 *Authors for correspondence: Yongbing Yuan (yyb@qau.edu.cn); Dongjie Jia (jiadongjie@qau.edu.cn).
13 *通讯作者:Yongbing Yuan ( yyb@qau.edu.cn); Dongjie Jia ( jiadongjie@qau.edu.cn).
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。

1

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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。
Keywords: apple, fruit coloration, carotenoid, R1-type MYB, abscisic acid (ABA), ABI5
关键词: 苹果 果实着色 类胡萝卜素 R1 型 MYB 脱落酸 (ABA) ABI5
Introduction 导言
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, 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 (