Elsevier

Plant Science

Volume 249, August 2016, Pages 46-58
Plant Science

Novel insight into the mechanism underlying light-controlled anthocyanin accumulation in eggplant (Solanum melongena L.)

https://doi.org/10.1016/j.plantsci.2016.04.001Get rights and content

Highlights

  • We functionally characterize SmCRY1, SmCRY2, SmCOP1 and SmHY5 from eggplant.

  • SmCRY1 and SmCRY2 interact with SmCOP1 in a blue-light-dependent manner.

  • SmCOP1 interacts with SmHY5 and SmMYB1.

  • SmHY5 and SmMYB1 both bind the promoters of SmCHS and SmDFR.

  • We provide a new working model underlying light-controlled anthocyanin accumulation.

Abstract

Eggplant is rich in anthocyanins, which are the major secondary metabolites and beneficial to human health. We discovered that the anthocyanin biosynthesis of eggplant cultivar ‘Lanshan Hexian’ was regulated by light. In this study, we isolated two blue light receptor genes, SmCRY1 and SmCRY2, and negative/positive anthocyanin regulatory factors SmCOP1 and SmHY5 from eggplant. In terms of transcript levels, SmCRY1, SmCRY2 and SmHY5 were up-regulated by light, while SmCOP1 was down-regulated. Subsequently, the four genes were functionally complemented in phenotype of corresponding mutants, indicating that they act as counterparts of Arabidopsis genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that SmCRY1 and SmCRY2 interact with SmCOP1 in a blue-light-dependent manner. It also obtained the result that SmCOP1 interacts with SmHY5 and SmMYB1. Furthermore, using yeast one-hybrid assay, we found that SmHY5 and SmMYB1 both bind the promoters of anthocyanin biosynthesis structural genes (SmCHS and SmDFR). Taken together, blue-light-triggered CRY1/CRY2-COP1 interaction creates the condition that HY5 and MYB1 combine with the downstream anthocyanin synthesis genes (CHS and DFR) in eggplant. Our finding provides a new working model by which light controls anthocyanin accumulation in eggplant.

Introduction

Anthocyanins, the largest subclass of water-soluble pigments synthesized via the flavonoid pathway, confer appealing color present in plants [1], [2]. Anthocyanins play important roles in a large range of plant functions, such as providing protection against UV radiation, low temperature, drought stress and pathogen attack [3], [4], [5], [6]. These pigments also help human to prevent several chronic diseases, such as cancer, cardiovascular disease, obesity and diabetes [7].

In the past few decades, the biosynthesis of anthocyanins was characterized in model plants, like Arabidopsis thaliana [8]. The biosynthesis process is catalyzed stepwise by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In addition, these structural genes are under the regulation of several transcription factors. To date, R2R3-MYB, basic-helix-loop-helix (bHLH), and WD Repeat Proteins (WDR), have been found to form a MYB-bHLH-WDR (MBW) complex to activate anthocyanin biosynthesis-related genes [9], [10]. The WDR proteins are thought to act as a bridge to stabilize the MBW complex, and are not thought to bind DNA [11]. The bHLH of the MBW complex does not limit anthocyanin accumulation, and they are involved in the determination of epidermal cell fate [12]. Recent studies have shown that the R2R3-MYB transcription factors, which directly affect the expression levels of the structural anthocyanin biosynthesis genes, play a central role in anthocyanin biosynthesis regulation [13], [14]. In Arabidopsis, AtMYB75, AtMYB90, AtMYB113 and AtMYB114, are essential for anthocyanin biosynthesis [8], [15]. A single AtMYB75, knockout mutant is sufficient for loss of anthocyanins in seedlings [15].

Light is an essential environmental factor for plant growth and development. Previous studies have shown that light can increase the anthocyanin concentration, especially in fruit skin [16], [17], [18]. There are several photoreceptors for perceiving light signal, including the blue/ultraviolet-A (320–500 nm)-light-absorbing cryptochromes (CRYs) [19]. Higher plants generally have two cryptochromes with clear functional diversification. In Arabidopsis, CRY1 specializes mostly in responses to high light and CRY2 mediates low light responses [20]. Cryptochromes regulate photomorphogenic development by suppression of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) activity directly or in association with SPA1 [21], [22], [23], [24]. Downstream of the photoreceptors, RING-finger type protein COP1 acts as a ubiquitin E3 ligase responsible for targeting several photomorphogenesis-promoting transcription factors, such as ELONGATED HYPOCOTYL 5 (HY5), HYPOCOTYL HOMOLOG (HYH), LONG AFTER FAR-RED LIGHT 1 (LAF1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1), and subsequently mediates their ubiquitination and degradation via the 26S proteasome pathway [25], [26], [27], [28]. HY5 is a basic leucine zipper transcription factor (bZIP) that binds directly to the promoters of light-inducible genes, such as anthocyanin structural genes, promoting their expression and photomorphogenic development [29], [30], [31], [32]. Recently, MdCOP1 was shown to interact with MdMYB1 that results subsequently in dark-induced degradation of MdMYB1 and in reduced anthocyanin accumulation in the peel of apple [33]. A similar mechanism for COP1-mediated control of an MYB transcription factor was recently reported to be present in Arabidopsis [34].

Eggplant (Solanum melongena L.) is an agronomically important vegetable, and is cultivated and consumed in many countries [35]. Eggplant cultivars with dark purple skin receive more attention for rich in anthocyanin, with concentrations as 2.34- and 7.08- fold more than grape (Vitis vinifera) and onion (Allium cepa) [36]. Due to the capacity of oxygen radical scavenging brought by anthocyanin included in fruit peel, eggplant is ranked as a member of the top ten vegetables [37]. Despite its commercial importance, few research efforts have been devoted to anthocyanin analysis of eggplant. The anthocyanin synthesis of some eggplant varieties, such as ‘Lanshan Hexian’, is completely regulated by light.

In this study, we isolate two blue light receptor genes, SmCRY1 and SmCRY2, and two photomorphogenesis transcriptional regulatory genes, SmCOP1 and SmHY5 from eggplant. Complementation assays using Arabidopsis mutants are performed to verify the four genes function. Then, we demonstrate that SmCRY1 and SmCRY2 interact with SmCOP1 in a blue-light-dependent manner, and SmCOP1 interact with SmHY5 and SmMYB1. Furthermore, we discover that SmHY5 and SmMYB1 both bind the promoters of anthocyanin biosynthesis structural genes. Taken together, our results suggest a light-induced working model that regulated an anthocyanin biosynthesis in eggplant.

Section snippets

Plant materials and treatments

The eggplant cultivar ‘Lanshan Hexian’ was grown in the horticultural farm of Shanghai Jiao Tong University, Shanghai, China. Samples of roots, stems, leaves, petals, peel and flesh from the same plants were collected for determining tissue-specific gene expression. The fruit bagging experiment included three treatments: bagging, where paper bags covered the sepals after full bloom; debagging, for which bags were removed after bagged 14 days at 8:00; and control, with fruits not being bagged at

Effect of bagging and debagging treatment on fruit color

As shown in Fig. 1, the fruits displayed white peel when they were bagged. Anthocyanins were not detected in the bagged fruit. After the bags removed, the sun-exposed fruits gradually became purple, and anthocyanin levels were gradually increased in the sun-exposed peel, suggesting that light controls anthocyanin biosynthesis in eggplant.

Isolation and sequence analysis

Since light controls anthocyanin biosynthesis in eggplant, we isolated SmCRY1, SmCRY2, SmCOP1 and SmHY5 by homologous cloning. Sequence data were deposited in

Discussion

In Arabidopsis, anthocyanins biosynthesis is a feature of light-grown plants and does not occur in dark-grown plants [33]. The reason is that COP1 acts as a central switch in light signal transduction by interacting with upstream light receptors and downstream target proteins [53], [54]. Our study also discovers that the anthocyanin biosynthesis of eggplant is completely regulated by light (Fig. 1). There are three types of light receptors: the blue light receptors cryptochromes CRY1 and CRY2,

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31471870), National Science Foundation for Young Scholars of China (31301770) and Shanghai Seed Industry of Agricultural Science Project (2013, No. 5).

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