Light-specific transcriptional regulation of the accumulation of carotenoids and phenolic compounds in rice leaves

ABSTRACT

Carotenoids and phenolic compounds are important subgroups of secondary metabolites having an array of functional roles in the growth and development of plants. They are also major sources for health and pharmaceutical benefits, and industrially relevant biochemicals. The control of the biosynthesis of these compounds depends mainly on the quality and quantity of different light sources. Thus, to unravel their light-specific transcriptional regulation in rice leaves, we performed promoter analysis of genes upregulated in response to blue and red lights. The analysis results suggested a crosstalk between different phytohormones and the involvement of key transcription factors such as bHLH, bZIP, MYB, WRKY, ZnF and ERF [jasmonic acid inducible], in the regulation of higher accumulation of carotenoids and phenolic compounds upon blue light. Overall, the current analysis could improve our understanding of the light-specific regulatory mechanism involved in the biosynthesis of secondary metabolites via possible critical links between different TFs in rice leaves.

Abbreviations

ABA

abscisic acid

GA

gibberellic acid

JA

jasmonic acid

LHCB

light-harvesting chlorophyll a/b-binding

TF

transcription factor

Plant carotenoids are involved in both photosynthesis and photoprotection, providing substrates for the biosynthesis of hormones.¹ They act as accessory pigments in the “Light Harvesting Complex” and then transfer the absorbed energy to chlorophyll a during photosynthesis.² Similarly, plant phenolic compounds are highly diverse group of secondary natural metabolites with an extremely large structural diversity.³ Their biological functions include phytoalexins, ultraviolet light protectants, pigments, signaling molecules, and mechanical strength (cell wall components). They can modulate essential physiological processes such as transcriptional regulation, membrane permeability, signal transduction, and vesicle trafficking. Hence, both carotenoids and phenolic compounds play multiple roles at different stages and aspects of plant life cycle mainly under biotic and abiotic stress conditions, plant reproduction, and diversity in flowers. Although the biosynthesis of these compounds has been extensively studied, the information on the light-driven regulatory mechanism is relatively limited. In our recently published paper,⁴ we have already discussed that in rice leaves, the highest and lowest accumulation of carotenoids and total phenolic in rice leaves, under blue light and dark conditions, respectively, following the order of blue>white>green>red>dark (carotenoids) and blue>white>red>green>dark (total phenolics). We also found a very divergent gene expression pattern in leaves grown in blue and red lights, which was consistent with the observed phenotypes. Since we hypothesized that the relative amount of blue and red lights are the major signaling factors to enhance the metabolic efficiency of rice plants, we aimed to study the transcriptional regulatory mechanism involved in the biosynthesis of carotenoids (α- and β-carotene, lutein) and phenolic compounds in rice leaves in response to these lights. To do so, we performed an ab initio analysis of cis-regulatory information content of the genes associated with the reporter metabolites.

Transcriptional regulation of carotenoids accumulation

The integration of the cis-element enrichment analysis with the gene expression data suggests the involvement of a number of transcription factors (TFs) for the transcriptional regulation of the higher accumulation of carotenoids in rice leaves in response to blue light. G-box-like elements ‘CACGTG’ associated with bZIP TFs and bHLH TFs were identified in the promoters of upregulated genes. In addition, the upregulation of several bZIP and bHLH TF genes such as, Os12g0156200 (DNA-binding factor of bZIP class), Os03g0741100 (basic helix-loop-helix dimerization region bHLH domain-containing protein) and Os01g0286100 (basic helix-loop-helix dimerization region bHLH domain-containing protein) further associates the regulatory roles of these TFs in the accumulation of carotenoids with the potential involvement in plant hormone signaling as suggested earlier (Fig. 1).^4-6 The promoter analysis results also suggest the role of bZIP TF as a positive regulator, and bHLH TFs particularly PIFs as negative regulators of chlorophyll and carotenoid accumulation.⁷ Among the bZIP TFs, HY5 is a strong antagonist of PIF and functions as an activator of genes for chlorophyll and carotenoid accumulation upon blue light signaling. Hence, in rice leaves both PIFs and HY5 seem to regulate an activation-suppression transcriptional control.⁸ HY5 has also been shown to regulate plant hormones such as gibberellic acid (GA) and abscisic acid (ABA) signal transduction. Overall, HY5 TF not only acts as a regulator in biosynthesis of carotenoids but also as a crosstalk between ABA and GA, and interaction between light and phytochrome signaling (Fig. 1).^9,10

Figure 1.

The proposed model of rice showing transcriptional regulation of biosynthesis of carotenoids and phenolic compounds in response to blue and red lights. The solid lines represent the findings which are supported by previous studies while the dotted lines are the hypothetical links proposed based on our analysis results.

The accumulation of carotenoids is also closely linked to the intracellular levels of GA and ABA. Biosynthesis of carotenoids and GA share a common biosynthetic precursor, i.e. geranylgeranyl diphosphate, and later carotenoids provide precursor for ABA biosynthesis.¹¹ Hence, the significant enrichment of ABRE-like, G-box-like, MYB-box-like, Pyrimidine-box-like, GARE-like elements in the upregulated genes and the upregulation of MYB and bZIP TF genes such as Os02g0685200 (Myb, DNA-binding domain containing protein), Os02g0706400 (Myb, DNA-binding domain containing protein) and Os12g0156200 (DNA-binding factor of bZIP class) in blue light indicates higher biosynthesis of ABA as well as GA. These results explain the positive feedback inhibition mediated by ABA to control stomatal conductance for chloroplast movement and photosynthesis. Since ABA is synthesized downstream of carotenoids, induction of carotenoid biosynthetic genes are also linked to the increased level of ABA. Moreover, the enrichment of PIF specific cis-elements supports the view that these TFs can also elevate the intracellular ABA levels by precisely promoting the expression of the ABA biosynthetic genes, and repressing the expression of the ABA catabolic genes.^12,13 Taken together, our results provide a link between the accumulation of higher carotenoids with higher concentration of both chlorophyll and ABA,¹⁴ and possible transcriptional regulation by TFs such as MYBs, bZIPs and bHLHs (PIFs) (Fig. 1). Additionally, the presence of W-box-like elements associated with a number of WRKY family TFs and the upregulation of Os02g0462800 (WRKY TF42 [TF WRKY02]) in blue light appear in balancing the positive function of the light-harvesting chlorophyll a/b-binding (LHCB). ABA is normally required in high level for the full expression of different LHCB proteins and then ABA-responsive WRKY-domain TF which represses LHCB expression acts to balance the positive function of the LHCBs.¹⁵ Therefore, in blue light treatment, ABA could act as an inducer to fine-tune LHCB expression in rice leaves and high level of carotenoids supports in activating photosynthesis and photoprotection. Higher biosynthesis of GA in both blue and red light treatment agrees with our identification of a number of GA response elements associated with MYB TF which are involved in the regulation of the biosynthesis of α- and β-carotene, and lutein. On the other hand, occurrence of ERE-like elements associated with a number of AP2/ERF TFs in both blue and red light possibly be acting as negative regulators in carotenoid accumulation which needs further experimental validation.¹⁶

Transcriptional regulation of phenolic compounds accumulation

We have identified several types of TFs that regulate the biosynthesis of different phenolic compounds. Of them, the roles of R1 MYB, R2-MYB, R2R3-MYB TFs in regulation of the synthesis of different phenylpropanoid-derived compounds have been extensively studied in many plants such as Arabidopsis, maize, rice, soybean and other plants.¹⁷ In the current analysis, the metabolic genes associated with the reporter metabolites were significantly enriched with putative cis-elements such as MYB-box-like, MYB-box related-like, GARE-like and pyrimidine-box-like associated with the different types of R1MYB, R2MYB and R2R3 MYB in response to both blue and red lights. The enrichment of these elements is supported by the upregulation of Os02g0685200 and Os02g0706400 in blue light, signifying their role in the regulation of the synthesis of different phenolic compounds. Moreover, presence of G-box-like elements associated with bHLH TF provides evidence to support the view that in blue light, there is possible interaction of MYB with bHLH TFs to accumulate more phenolic compounds. Higher accumulation of ABA, high enrichment of W-box-like-elements associated with WRKY TFs together with the upregulation of Os02g0462800 (WRKY transcription factor 42 [Transcription factor WRKY02]) in blue light supports the role of ABA-responsive WRKYs in the regulation of the biosynthesis of phenolic compounds (Fig. 1).^18-22 Our analysis also shows the involvement of ZnF TFs (ZCT1, ZCT2 and ZCT3) in the regulation of the biosynthesis of phenolic compounds which needs further validation. In addition to the high enrichment of putative cis-elements associated with TFs such as, bHLH, bZIP, MYB, WRKY and ZnF, genes encoding members of these TF families also showed an upregulation. These genes are known for their roles against abiotic and biotic stresses, so it is possible that these TFs have a general ability to regulate production of phenolic compounds. Presence of JARE-like the motifs associated with ERF TF family [jasmonic acid (JA) induced] and upregulation of ERF TF genes provide support in favor of the involvement of JA-mediated biosynthesis of phenolic compounds. However, experimental validation is required to support our hypothesis.

Overall, our results suggest the potential involvement of a number of TFs and a cross talk between different phytohormones such as GA, ABA and JA for the regulation of carotenoids and phenolic compounds in rice leaves in response to blue and red lights.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Synthetic Biology Initiative of the National University of Singapore (DPRT/943/09/14) and the Next-Generation BioGreen 21 Program of the Rural Development Administration, Republic of Korea (Systems and Synthetic Agrobiotech Center; grant no. PJ01109405).