Increasing flavonoid contents of tomato fruits through disruption of the SlSPL-CNR, a suppressor of SlMYB12 transcription activity.
Increasing flavonoid contents of tomato fruits through disruption of the SlSPL-CNR, a suppressor of SlMYB12 transcription activity.
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DOI：
10.1111/pbi.14214
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发表时间：
2024-02
影响因子：
13.8
通讯作者：
Qu, Guiqin
中科院分区：
文献类型：
--
作者：
Zhou, Leilei;Sun, Zongyan;Hu, Tingting;Chen, Di;Chen, Xue;Zhang, Qiaoli;Cao, Jiankang;Zhu, Benzhong;Fu, Daqi;Zhu, Hongliang;Qu, Guiqin
番茄果实营养丰富，是人类重要的食物来源。在果皮中，附着在果肉上的果皮组织产生功能性成分，包括实际上是混合多酚化合物的类黄酮。香精在确定果皮颜色中起着重要作用，并且被证明作为有利的亲水性抗氧化剂对人类健康具有重要的功能（Ballester等人，2010年）。然而，包括番茄在内的许多水果中内源类黄酮的含量往往不足，达不到保健的要求。因此，挖掘番茄类黄酮调控基因，培育富含类黄酮的番茄品种是番茄品质改良的重要目标。黄酮类化合物的生物合成来源于苯丙素类化合物合成途径，并通过一系列酶的组合转化，这些酶主要由黄酮类化合物生物合成基因控制。S1 MYB 12，一种R2 R3-MYB转录因子，通过广泛靶向和激活类黄酮生物合成基因的转录而被发现是类黄酮生物合成中的主调节因子（Adato等人，2009年）。SlMYB 12基因的破坏导致柚皮素查耳酮的积累减少和粉红色番茄果实（Ballester et al.，2010），表明SlMYB 12的调节可能是控制番茄皮中类黄酮生物合成的有效策略。除了其在介导类黄酮含量中的重要作用外，SlMYB 12转录活性的分子基础在很大程度上仍然未知。番茄成熟抑制蛋白（SBP box protein Colorless Non-ripening，SlSPL-CNR）是番茄成熟调控转录因子中的一个。虽然我们最近发现由CRIPSR/Cas9系统产生的slcnr突变体的果实可以变成全红色，这与不能成熟的原始Cnr突变体不同（Gao等人，2019年）。在此，我们进一步研究了SlCNR蛋白完全缺失的slcnr突变体（slcnr-16、slcnr-22和slcnr-23）的果实成熟特性（图S1），发现它们仅表现出成熟起始延迟约3天和番茄红素含量轻微降低（图S2）。这些结果表明，SlCNR不是果实成熟的主调节因子，其突变不会改变整个成熟过程。使用抗-SlCNR多克隆抗体的蛋白质印迹分析揭示了SlCNR蛋白在果实中主要表达（图1a）。此外，果皮组织比果肉含有明显更丰富的SlCNR蛋白（图1b），表明SlCNR在调节果皮发育中可能具有重要的功能。有趣的是，slcnr突变体的果实在成熟过程中表现出黄橙色外观，而野生型似乎迅速变红，并且前者比后者具有更深的黄色果皮组织（图1c）。在Br和Br+ 7野生型果实之间鉴定了总共2165个差异表达基因（DEG;倍数变化≥ 2且P值< 0.05），其中8%（187）和92%（1978）在Br+ 7果实中分别上调和下调（图S4 a，表S1）。在slcnr-23突变体中，在Br和Br+ 7果实之间鉴定了1603个DEG，其中21%（341个）在Br+7果实中上调，78%（1162个）在Br+ 7果实中下调（图S4 b，表S2）。与野生型相比，DEG分别为362和573。
Fruits of tomato (Solanum lycopersicum) are rich in nutrients and generally served as critical resources for human diet. In fruit pericarps, the peel tissues adhering to the flesh produce functional components including flavonoids that are indeed mixed polyphenolic compounds. Flavonoids play an essential role in determining peel colour and are proven to be functionally important for human health as favourable hydrophilic antioxidants (Ballester et al., 2010). However, the content of endogenous flavonoids in numerous fruits including tomato tends to be inadequate and fails to meet the requirement of health benefits. Therefore, digging in essential flavonoid-regulated genes and further breeding of tomato fruits rich in flavonoids is a significant target for tomato improvement in quality. Flavonoid biosynthesis is derived from the phenylpropanoid pathway and undergoes combinatorial transformations by a series of enzymes, which are mainly controlled by flavonoid biosynthetic genes. SlMYB12, a R2R3-MYB transcription factor, was uncovered to be a master regulator in flavonoid biosynthesis by extensively targeting and activating the transcription of flavonoid biosynthetic genes (Adato et al., 2009). Disruption of SlMYB12 gene resulted in the decreased accumulation of naringenin chalcone and a pink-coloured tomato fruit (Ballester et al., 2010), indicating that modulation of SlMYB12 could be an efficient strategy in controlling flavonoid biosynthesis in tomato peel. Apart from its significant role in mediating flavonoid content, the molecular basis underlying the transcriptional activity of SlMYB12 remains largely unknown. The tomato SBP-box protein Colorless Non-ripening (SlSPL-CNR, referred as to SlCNR) was early cloned and studied as a master ripening transcription factor. While we recently found that fruits of the slcnr mutants generated by CRIPSR/Cas9 system can turn to full red, which are distinct from the original Cnr mutant that fails to ripen (Gao et al., 2019). Herein, we further investigated the maturity characteristics in fruits of the slcnr mutants (slcnr-16, slcnr-22 and slcnr-23) with the complete deficiency of SlCNR protein (Figure S1) and found that they only exhibited an approximately 3-day delay in ripening initiation and a slight decreased lycopene content (Figure S2). These results demonstrated that SlCNR is not a master regulator of fruit ripening, and its mutation does not change the overall ripening process. Western blot analysis using the anti-SlCNR polyclonal antibody revealed that SlCNR protein dominantly expresses in fruits (Figure 1a). Moreover, the peel tissues harbour obviously more abundant SlCNR protein than the flesh (Figure 1b), indicating that SlCNR may be functionally important in modulating the development of fruit peel. Intriguingly, fruits of the slcnr mutants exhibited a yellow-orange colour appearance during the ripening, while the wild-type appeared to red rapidly, and the former harboured a deeper yellow-coloured peel tissues than the later (Figure 1c).To further investigate the regulation role of SlCNR on fruit colour, a comparative transcriptomic analysis was performed with high Pearson correlation coefficients (Figure S3). A total of 2165 differentially expressed genes (DEGs; fold change≥ 2 and P value< 0.05) were identified between Br and Br+ 7 wild-type fruits, of which 8%(187) and 92%(1978) were up-regulated and down-regulated in Br+ 7 fruits, respectively (Figure S4a, Table S1). In slcnr-23 mutant, 1603 DEGs were identified between Br and Br+ 7 fruits, of which 21%(341) were upregulated and 78%(1162) were down-regulated in Br+ 7 fruits (Figure S4b, Table S2). Compared with the wild-type, 362 and 573 DEGs were …
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