Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco

Abstract

The interaction between noncoding endogenous target mimicry (eTM) and its corresponding microRNA (miRNA) is a newly discovered regulatory mechanism and plays pivotal roles in various biological processes in plants. Tobacco (Nicotiana tabacum) is a model plant for studying secondary metabolite alkaloids, of which nicotine accounts for approximately 90%. In this work, we identified four unique tobacco-specific miRNAs that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways and an eTM, novel tobacco miRNA (nta)-eTMX27, for nta-miRX27 that targets QUINOLINATE PHOSPHORIBOSYLTRANSFERASE2 (QPT2) encoding a quinolinate phosphoribosyltransferase. The expression level of nta-miRX27 was significantly down-regulated, while that of QPT2 and nta-eTMX27 was significantly up-regulated after topping, and consequently, nicotine content increased in the topping-treated plants. The topping-induced down-regulation of nta-miRX27 and up-regulation of QPT2 were only observed in plants with a functional nta-eTMX27 but not in transgenic plants containing an RNA interference construct targeting nta-eTMX27. Our results demonstrated that enhanced nicotine biosynthesis in the topping-treated tobacco plants is achieved by nta-eTMX27-mediated inhibition of the expression and functions of nta-miRX27. To our knowledge, this is the first report about regulation of secondary metabolite biosynthesis by an miRNA-eTM regulatory module in plants.

Nicotine biosynthesis is regulated by two more layers: Small and long non-protein-coding RNAs

“…14 These studies demonstrated that the nicotine biosynthesis is regulated by 2 more layers distinct from previously known mechanisms. One layer is through a miRNAmediated gene silencing mechanism to degrade the transcripts of QPT2 by miRNA nta-miRX27 (Fig.…”

“…14 We discovered that the cleavage activity of nta-miRX27 was negatively regulated by nta-eTMX27 leading us to propose that another layer of control is exerted over QPT2 expression and nicotine biosynthesis via an eTM. An eTM acting as a decoy for the microRNA to buffer its target gene is a newly emerging regulation mechanism on gene expression.…”

“…The mature nta-miRX27 that we identified in our study 14 is derived from a 131 bp precursor having 21 nucleotides, which fits a typical miRNA length of 20-22 nucleotides. 15 By prediction, the nta-miRX27 could bind QPT2 at 2 sites located at exon 2 and intron 8 with targeting scores of 4.5 and 2.5, respectively.…”

“…20,21 Topping by removal of the plant/flower head has both transient wounding effect as well as lasting low auxin effect on QPT2 expression and nicotine production. So far, the expressions of nta-miRX27 13,14,28 and nta-eTMX27 14 respond to the topping treatment. Whether or not wounding and auxin affect ntamiRX27 and nta-eTMX27 expressions remains to be determined.…”

MicroRNA, a new target for engineering new crop cultivars

“…For example, Li and colleagues (2015) identified 4 tobacco-specific miRNAs targeting nicotine biosynthesis pathway; regulating these miRNAs affect nicotine contains in tobacco and further affect tobacco quality. 25 Using deep sequencing technology, Boke and colleagues (2015) also identified several miRNAs regulating alkaloid biosynthesis. 26 Phytosterols are membrane components or precursors for brassinosteroid 27 biosynthesis; a recent study show that miR1848 regulates phytosterol biosynthesis by targeting obtusifoliol 14a-demethylase gene OsCYP51G3 in rice.…”