Mellowed Yellow: WHITE PETAL1 Regulates Carotenoid Accumulation in Medicago Petals

Author: Philip Carella; Affiliations: University of Cambridge (剑桥大学): Cambridge, UK

Carotenoids are tetraterpenoid (C40) lipophilic compounds that are widely distributed in nature and play key roles in pigmentation, photosynthesis, and development (Nisar et al., 2015). Detailed biochemical and genetic analyses have uncovered the carotenoid biosynthetic pathway, which generates a diverse array of molecules that are either colorless or impart red (lycopene), orange (carotene), or yellow (xanthophyll) pigmentation in distantly-related photosynthetic organisms.

类胡萝卜素是四萜类（C40）的亲脂性化合物，在自然界广泛分布，并在色素沉着、光合作用以及发育过程中发挥着关键作用。详细的生化和遗传分析已经发现了类胡萝卜素的生物合成途径，该途径在不同的、远缘光合生物中产生了各种各样的分子，这些分子即包括了无色的化合物，也包括了有色的，比如红色的番茄红素、橙色的胡萝卜素以及黄色的叶黄素等。

Despite a strong mechanistic understanding of carotenoid biosynthesis, relatively little is known about how this pathway is transcriptionally regulated (reviewed in Stanley and Yuan 2019). Insight into carotenoid regulation is provided by Meng et al. (2019), who screened Medicago truncatula insertion mutants for the loss of carotenoid-derived pigment accumulation in petals. This identified two allelic mutants, white petal1-1 (wp1-1) and wp1-2, displaying pale white petal coloration in comparison to the yellow petals of wild-type plants. Biochemical analyses confirmed that wp1 mutant petals failed to accumulate carotenoids associated with yellow pigmentation, including esterified lutein, lutein epoxide, and violaxanthin.

尽管我们对于类胡萝卜素的生物合成已经有了很深的理解，但对该途径如何受到转录调控还不是很清楚。 Meng等人最近发表的一篇文章为我们提供了对类胡萝卜素转录调节的最新见解，他们筛选了苜蓿插入突变体以寻找是否存在花瓣中类胡萝卜素衍生色素积累缺陷的突变体。作者通过这种方法鉴定了两个等位基因突变体，白色花瓣wp1-1和wp1-2，与野生型苜蓿的黄色花瓣相比，这两个突变体的花瓣呈现出淡白色。生化分析证实，wp1突变体的花瓣未能积累黄色色素沉着相关的类胡萝卜素，包括酯化的叶黄素、叶黄素环氧化物以及紫黄质。

Molecular cloning revealed that WP1 encodes a R2R3-MYB transcription factor belonging to the subgroup 6 class of MYBs that typically regulate anthocyanin biosynthesis in flowering plants. Genetic complementation of wp1 mutants with a genomic WP1 sequence fused to GFP (green fluorescence protein) fully restored carotenoid-derived yellow pigmentation in WP1-GFP/wp1 petals (see Figure). Moreover, expression analysis demonstrated that WP1 transcripts are most abundant in floral organs, consistent with its role in floral pigment accumulation.

分子克隆表明，WP1编码一个R2R3-MYB转录因子家族第六亚家族的成员，这类转录因子通常在开花植物中调控花青素的生物合成。通过WP1-GFP融合蛋白的wp1突变体遗传互补试验能够完全恢复WP1-GFP/wp1中花瓣的类胡萝卜素衍生黄色色素沉着。此外，基因表达分析显示WP1转录本在花器官中最丰富，这与其在花瓣中的色素积累作用相一致。

To further explore the role of WP1 in Medicago, the authors assessed the expression levels of carotenoid biosynthesis genes in wp1 relative to wild-type petals. As expected, functional WP1 was required for the full extent of carotenoid biosynthesis gene expression. Further characterization of the WP1 protein revealed that its C-terminus was required for strong transcriptional activation. Next, the authors queried the promoters of WP1-dependent carotenoid biosynthesis genes for conserved MYB-binding elements to identify putative WP1 binding sites important for floral pigmentation. Using chromatinimmunoprecipitation (ChIP)-based DNAbinding experiments and heterologous transactivation assays, the authors confirmed that WP1 directly binds to the promoters of the MtLYCe (lycopene ε–cyclase) and MtLYCb (lycopene β-cyclase) carotenoid biosynthesis genes and induces their expression.

为了进一步研究WP1在紫花苜蓿中的作用，作者进一步评估了wp1突变体中类胡萝卜素生物合成基因相对于野生型花瓣的表达水平变化。不出所料，功能性的WP1对于类胡萝卜素生物合成相关基因的充分表达是必要的。 进一步对于WP1蛋白的鉴定发现其C端是强转录激活所必需的。接着，作者查询了WP1依赖型类胡萝卜素生物合成基因启动子区中保守的MYB结合元件，用来鉴定对于花瓣色素沉着必要的WP1可能的结合位点。通过染色质免疫沉淀（ChIP）的DNA结合实验以及异源反式激活试验，作者确定了WP1能够直接结合到番茄红素ε-环化酶MtLYCe和番茄红素β-环化酶MtLYCb类胡萝卜素合成基因的启动子上，并诱导这两个基因的表达。

In Medicago, several R2R3-MYBs interact with the bHLH (basic helix-loop-helix) transcription factor TRANSPARENT TESTA8 (MtTT8) and the WDR (WD-repeat domain) protein MtWD40-1, forming a MYB-bHLHWDR (MBW) complex that regulates anthocyanin biosynthesis (Li et al., 2016). Intriguingly, both mttt8 and mtwd40-1 mutants displayed pale yellow coloration and decreased lutein levels relative to wild-type controls, which suggests that a WP1-containing MBW complex contributes to carotenoid regulation. Consistent with this idea, WP1-based DNA-binding and transactivation assays performed with and without MtTT8 and MtWD40-1 demonstrated that the presence of a WP1-MtTT8-MtWD40-1 complex results in the maximal activation of carotenoid biosynthesis. Meng and colleagues revealed an additional role for WP1-MtTT8-MtWD40-1 in regulating anthocyanin accumulation in the pigmented veins radiating from the base of the vexillum (banner) petal. Collectively, this work identifies WP1 as a key regulator of carotenoid accumulation in Medicago petals. That WP1 functions within an MWB regulatory module to control carotenoid and anthocyanin accumulation in discrete areas of the petal provides a novel framework to investigate the evolutionary history of plant secondary metabolism and floral pigmentation.

在苜蓿中，有几个R2R3-MYB转录因子可以与bHLH转录因子MtTT8及WDR蛋白MtWD40-1互作，形成MYB-bHLH-WDR，即MBW复合物，从而调控花青素的生物合成。有趣的是，相对于野生型，mttt8和mtwd40-1突变体的花瓣均呈现出淡黄色、且叶黄素水平降低，这表明含WP1的MBW复合物作用于类胡萝卜素的调节。与此一致的是，在有和没有MtTT8和MtWD40-1的情况下进行的基于WP1的DNA结合和反式激活试验表明，存在WP1-MtTT8-MtWD40-1复合物情况下会最大程度激活类胡萝卜素的生物合成。 Meng和同事揭示了WP1-MtTT8-MtWD40-1在调控由花瓣根部辐射出的有色叶脉中花青素积累此外的其他功能。总的来说，这项工作确定WP1是紫花苜蓿花瓣中类胡萝卜素积累的一个关键调控因子。WP1在MWB复合物调节模块中发挥功能，控制花瓣离散区域中的类胡萝卜素和花青素积累，为研究植物次生代谢和花色的演化史提供了一个新的框架。

通讯：Philip Carella (https://www.slcu.cam.ac.uk/people/carella-philip)

个人简介：加拿大麦克马斯特大学，博士。

doi: https://doi.org/10.1105/tpc.19.00728

Journal: Plant Cell

First Published: September 27, 2019