Short communication
Ethylene mediates repression of anthocyanin accumulation in black rice pericarps in the absence of light

https://doi.org/10.1016/j.jplph.2019.02.011Get rights and content

Abstract

The antioxidant properties of black rice are attributed to the high anthocyanin content in the pericarp. Light-dependent regulation of anthocyanin biosynthesis and the associated regulatory genes have been extensively studied in many plant species, including rice. Light is considered indispensable for anthocyanin accumulation in plants. Here, we report that anthocyanin biosynthesis and accumulation in the dark is negatively regulated by ethylene, as the ethylene biosynthesis inhibitor aminoethoxyvinylglycine hydrochloride (AVG)-treated samples in the dark exhibited significantly higher transcript levels of biosynthesis genes, including CHI, DFR, ANS, ANR, F3H, F’3H, CHS and UGFT, compared to untreated controls. Upregulation of these biosynthesis genes was accompanied by simultaneous de-repression of the R2-R3 domain and bHLH-containing transcription factors Kala3 and Kala4 at the transcript level. Additionally, bHLH152, which shows high similarity to Arabidopsis PIF3, is involved in the regulation of anthocyanin. These findings highlight the role of ethylene in modulating tissue-specific regulation of anthocyanin biosynthesis genes in the dark.

Introduction

Anthocyanin is a water-soluble flavonoid (Shen et al., 2009) residing on the pericarp, seed coat and aleurone layer of rice seeds (Sompong et al., 2011; Ryu et al., 1998a; Takashi et al., 2001). Anthocyanins are a source of antioxidants that have the capacity to inhibit highly reactive free radicals, which can damage the cell (Adom and Liu, 2002). Black rice constitutes a class of specialty rice varieties that are glutinous, packed with high levels of nutrients, and cultivated in many countries across Asia. The black coloration of the kernel pericarp (outer part) is attributed to a class of flavonoid pigments known as anthocyanin (Carey et al., 2004). Black rice is also considered by many as a panacea for food-related diseases because of its high nutritive value, curative effects and beneficial properties of flavonoids that not only act as antioxidant (Reddy et al., 1996; Ryu et al., 1998; Saitoh et al., 2004; Shao et al., 2011) and anti-inflammatory (Sakamoto et al., 2001) agents but have also been linked to anti-carcinogenic properties (Shao et al., 2011; Shih et al., 2008).

Early biochemical studies have reported cyaniding-3-O-glucoside (C3G) as the primary fraction of anthocyanin in addition to Melvin-3 glycosides and peonidin-glucoside (P3G) (Cone et al., 1993). Black or purple pericarp traits occur due to mutation events in certain key regulatory genes during the course of crop evolution and domestication. While most cultivated rice (Oryza sativa) varieties have white grains, wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins because of the presence of a defective allele in the bHLH domain containing the Rc gene. Wide-scale comparative genomic analysis of white-grained landraces such as Nipponbare has led to the identification of the causal allele of Kala4. Further confirmation of the presence of this allele that was restricted to a genomic fragment from tropical japonica in black-trait indica varieties implies introgression of the trait due to occurrence of natural crossbreeding over the course of evolution (Oikawa et al., 2015). The biosynthesis of anthocyanins is mediated by multiple enzymes in the phenylpropanoid pathway (Koes et al., 2005), and their coding genes are grouped into two classes, namely, early and late biosynthesis genes (Tanaka et al., 2008), whose regulation is coordinated by discrete phytohormone signalling pathways through their actions on a subgroup of R2-R3 MYB, bHLH and WD40 domain-containing transcription factors. While cytokinin, abscisic acid (ABA) and jasmonic acid (JA) reportedly induce anthocyanin, GA, ethylene, and brassinosteroid repress its biosynthesis in Arabidopsis (Peng et al., 2011). Early biosynthetic genes, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H) and flavonol synthase (FLS), are preferentially activated by R2R3-MYB regulatory genes, such as MYB11, MYB12 and MYB111 (Stracke et al., 2007). In contrast, late biosynthesis genes, including dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP-glucose:flavonoid 3-O-glucosyl transferase (UF3GT), are primarily regulated by a MYB-bHLH-WD40 (MBW) transcription complex, a ternary transcriptional factor complex consisting of R2R3 MYB-activators, bHLH activators and the WD-repeat protein TTG1 (Gonzalez et al., 2008; Matsui et al., 2008; Petroni and Tonelli, 2011). Rice R2-R3 MYB transcription factor Kala4, along with the bHLH domain-containing transcription factor Kala3, regulates the accumulation of anthocyanin in the pericarp through their actions on anthocyanin biosynthesis genes (Oikawa et al., 2015; Park, 2012).

Light is the primary external factor that modulates the intensity of anthocyanin and is considered indispensable for anthocyanin induction because no accumulation was reported under dark conditions. Sugar and cytokinin are known to enhance the accumulation of anthocyanin in light but failed to accumulate any pigment in dark-grown maize mesocotyls (Piazza et al., 2003; Jeong et al., 2010). The absence of anthocyanin pigmentation in the dark has been attributed to the COP1/SPA complex (Chen et al., 2006), primarily through ubiquitination and subsequent proteolytic degradation of activators of the light response, such as the transcription factors HY5 and HFR1 by the 26S proteasome (Hoecker, 2005; Yi and Deng, 2005). However, while ethylene suppression of anthocyanin accumulation in light is mediated through leucine-zipper transcription factor HY5 acting as a downstream component of phytochrome (PHY), cryptochrome (CRY), and UV-B (UVR8) photoreceptors (Jeong et al., 2010), cytokinin enhancement of anthocyanin accumulation could occur independently of HY5 in light conditions (Das et al., 2012). Pigmented flavonoids were previously used as phenotypic markers in many plant species and have proved to be an excellent model for elucidating the biochemical and genetic basis underlying tissue specificity. Transcriptome studies of genes regulating the tissue-specific accumulation of anthocyanin in the black rice pericarp have been performed (Kim et al., 2010). However, the influence of factors such as light, sugar, and phytohormones on the organ-specific regulation of anthocyanin biosynthesis in the immature black rice pericarp is not well understood. Thus, the present research demonstrates the organ-specific effect of ethylene on the biosynthesis and accumulation of anthocyanin in the dark using immature black rice pericarps. We have experimentally demonstrated that, in the dark, ethylene transcriptionally suppresses the Kala3 and Kala4 transcription factors that are positively associated with light-induced anthocyanin accumulation in developing black rice pericarps.

Section snippets

Plant material

Black rice was collected from the polyhouse at different developmental stages (6, 12 and 18 days after heading, DAH) and carefully dehulled for quantification of total anthocyanin content as described below. To evaluate the effect of light and hormones on anthocyanin accumulation, immature black rice seeds were collected at 6 DAH (Fig. 1A) and sterilized with 3% sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were placed in Petri dishes and subjected to

Suppression of anthocyanin accumulation in the dark is reversed by inhibition of ethylene biosynthesis

Sugar and light induce anthocyanin biosynthesis while ethylene represses anthocyanin biosynthesis in Arabidopsis at the vegetative stage (Das et al., 2012). The role of such factors in the tissue-specific anthocyanin regulation of developing black rice pericarps was determined. No pigmentation was observed in dark-incubated samples (immature black rice seeds) supplemented with or without exogenous sugar (90 mM sucrose) when compared to samples exposed to light (Fig. 1B). However, treatment of

Conclusion

In this study, we found high anthocyanin contents in the pericarp of black rice treated with AVG in the absence of light, which correlates with the corresponding gene being over-expressed as a result of the priming effect of biosynthetic and regulatory genes.

Light is the primary external factor that modulates the intensity of anthocyanin and is considered indispensable for anthocyanin induction because no accumulation was reported under dark conditions. Our results indicate the organ-specific

Funding

This work was supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India. Grant no. NER/AGRI/29/2015 (Group -5).

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    Present address: Swedish University of Agricultural Sciences, Box 101, 230 53, Alnarp, Lomma, Sweden.

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