Comparative Transcriptomic Analyses of Differentially Expressed Genes in Transgenic Melatonin Biosynthesis Ovine HIOMT Gene in Switchgrass.

Cong Guan,Yunwei Zhang,Xin Cui,Shan Yuan,Fuyu Yang,Sijia Liu,Yanhua Huang,Danyang Tian

doi:10.3389/fpls.2016.01613

Abstract

Melatonin serves pleiotropic functions in prompting plant growth and resistance to various stresses. The accurate biosynthetic pathway of melatonin remains elusive in plant species, while the N-acetyltransferase and O-methyltransferase were considered to be the last two key enzymes during its biosynthesis. To investigate the biosynthesis and metabolic pathway of melatonin in plants, the RNA-seq profile of overexpression of the ovine HIOMT was analyzed and compared with the previous transcriptome of transgenic oAANAT gene in switchgrass, a model plant for cellulosic ethanol production. A total of 946, 405, and 807 differentially expressed unigenes were observed in AANAT vs. control, HIOMT vs. control, and AANAT vs. HIOMT, respectively. Two hundred and seventy-five upregulated and 130 downregulated unigenes were detected in transgenic oHIOMT line comparing with control, including the significantly upregulated (F-box/kelch-repeat protein, zinc finger BED domain-containing protein-3) genes, which were potentially correlated with enhanced phenotypes of shoot, stem and root growth in transgenic oHIOMT switchgrass. Several stress resistant related genes (SPX domain-containing membrane protein, copper transporter 1, late blight resistance protein homolog R1A-6 OS etc.) were specifically and significantly upregulated in transgenic oHIOMT only, while metabolism-related genes (phenylalanine-4-hydroxylase, tyrosine decarboxylase 1, protein disulfide-isomerase and galactinol synthase 2 etc.) were significantly upregulated in transgenic oAANAT only. These results provide new sights into the biosynthetic and physiological functional networks of melatonin in plants.

Highlights

The last two steps are presumed as rate-limiting which are catalyzed by arylalkylamine N-acetyltransferase (AANAT)/serotonin N-acetyltransferase (SNAT) and HIOMT/aceylserotonin methyltransferase (ASMT) (Morton and Forbes, 1988; Byeon et al, 2015, 2016)
The overexpression of rice caffeic acid O-methyltransferase (COMT) exhibited upregulation of melatonin contents in transgenic rice plants, indicating that the N-acetylserotonin methyltransferase activity was required for melatonin biosynthesis (Byeon et al, 2015)
Transgenic HIOMT switchgrass exhibited significant promotion of growth comparing with empty vector (EV) (Figure 1; Supplementary Table 3)

Summary

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) was first discovered in vertebrates (Lerner et al, 1958), detected in higher plants (Dubbels et al, 1995; Hattori et al, 1995), and now is widely accepted of its distribution in all kingdoms, from prokaryotes to eukaryotes, from animals to plants (Manchester et al, 2000; Hardeland and Poeggeler, 2003; Simopoulos et al, 2005; Hardeland, 2015), and functions as a direct scavenger of reactive oxygen species (ROS), a mediator hormone of circadian rhythms and an activator of antioxidant enzymes (Arnao and HernándezRuiz, 2015; Reiter et al, 2015; Bai et al, 2015, 2016; Gao et al, 2016; Shi et al, 2016a). The involved regulations of the functional gene expression and physiological mechanism of melatonin biosynthesis and metabolic pathways remains poorly understood in plants (Hardeland, 2015; Zhang et al, 2014b). The classic pathway of melatonin biosynthesis comprises four steps beginning with tryptophan, firstly decarboxylation by tryptophan decarboxylase (TDC), N-acetylation by arylalkylamine N-acetyltransferase (AANAT) in animals/serotonin N-acetyltransferase (SNAT) in plants, and the final O-methylation by N-aceylserotonin methyltransferase (ASMT) in animal/hydroxyindole-O-methyltransferase (HIOMT) in plants (Tan et al, 2016). Genetic engineering modifications of coding genes for melatonin biosynthesis and metabolism enzymes were applied to alter the melatonin contents in transgenic rice, tomato, Arabidopsis thaliana, and Nicotiana sylvestris (Kang et al, 2010; Okazaki et al, 2010; Park et al, 2012; Zhang et al, 2012; Wang et al, 2014; Zuo et al, 2014)

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Results

Conclusion