Effects of overexpression of jasmonic acid biosynthesis genes on nicotine accumulation in tobacco

Abstract

Nicotine is naturally synthesized in tobacco roots and accumulates in leaves as a defense compound against herbivory attack. Nicotine biosynthesis pathway has been extensively studied with major genes and enzymes being isolated and functionally characterized. However, the molecular regulation of nicotine synthesis has not been fully understood. The phytohormone jasmonic acid (JA) mediates many aspects of plant defense responses including nicotine biosynthesis. In this study, five key genes (AtLOX2, AtAOS, AtAOC2, AtOPR3, AtJAR1) involved in JA biosynthesis from Arabidopsis were individually overexpressed, and a JA-Ile hydrolysis-related gene, NtJIH1, was suppressed by RNAi approach, to understand their effects on nicotine accumulation in tobacco. Interestingly, while transgene expression was high, levels of JA-Ile (the biologically active form of JA) were often significantly reduced.Meanwhile, nicotine content in these transgenic plants did not increase. The research revealed a tightly controlled JA signaling pathway and a complicated regulatory network for nicotine biosynthesis by JA signaling. K E Y W O R D Sjasmonic acid biosynthesis, jasmonic acid signaling, nicotine synthesis regulation, tobacco, transgene 1 | INTRODUCTION Tobacco (Nicotiana tabacum L.) is an important nonfood crop widely grown across the world due to its great economic value brought by the widespread usage of tobacco products (Davis & Nielsen, 1999).Nicotine is specifically synthesized in tobacco roots and accumulated in leaves as a defensive compound against herbivores because it causes a continual excitation of neurons and even paralysis or death of insects (Baldwin, Halitschke, Kessler, & Schittko, 2001). Thus, nicotine is used as an insecticide in agriculture practice (Davis & Nielsen, 1999). In medical applications, nicotine can be used to make smoking cessation devices (Wang et al., 2015) and is applied to treating Parkinson's disease and alleviating inflammatory bowel syndrome (Polosa, Rodu, Caponnetto, Maglia, & Raciti, 2013;Quik, O'Leary, & Tanner, 2008). Nicotine is a major type of alkaloids in tobacco plants, accounting for 90% of the total alkaloids. The rest 10% are mainly composed of anabasine, anatabine, and nornicotine (Saitoh, Nona, & Kawashima, 1985).Nicotine is composed of a pyridine ring and a pyrrolidine ring, synthesized from two separate branches as demonstrated in Fig-ure S1. The pyrrolidine ring is originated from arginine or ornithine while the pyridine ring is formed from quinolinic acid. Early studies reported that the putrescine methyltransferase (PMT) and quinolinic acid phosphoribosyltransferase 2 (QPT2) are the rate-limiting enzymes in the pyrrolidine branch and pyridine branch, respectively, because they had much lower enzyme activities than other enzymes wileyonlinelibrary.com/journal/pld3 | 1 in the two branches (Feth, Wagner, & Wagner, 1986;Saunders & Bush, 1979;Wagner & Wagner, 1985). The isoflavone reductase-like enzyme A622 and a berberine bridgelike (BBL) enzyme are proposed to be involved in...

Overexpression of Arabidopsis OPR3 in Hexaploid Wheat (Triticum aestivum L.) Alters Plant Development and Freezing Tolerance

“…Similar traits, such as faster growth, early senescence, and sensitivity to short-term freezing were also observed in Tr-15 (Figures S3–S5). Currently, we are unable to explain the mechanisms leading to a decrease in JA level upon overexpression of the AtOPR3 gene, but similar results have been previously obtained in several studies describing transgenic plants overexpressing jasmonates biosynthesis pathway genes [12,59]. Transgenic tobacco plants overexpressing few JA pathway genes from Arabidopsis, including AtOPR3 , had a decreased level of jasmonate–isoleucine conjugate, the most active metabolite of the pathway [59].…”

“…Currently, we are unable to explain the mechanisms leading to a decrease in JA level upon overexpression of the AtOPR3 gene, but similar results have been previously obtained in several studies describing transgenic plants overexpressing jasmonates biosynthesis pathway genes [12,59]. Transgenic tobacco plants overexpressing few JA pathway genes from Arabidopsis, including AtOPR3 , had a decreased level of jasmonate–isoleucine conjugate, the most active metabolite of the pathway [59]. Interestingly, the expression level of jasmonate-regulated genes involved in nicotine biosynthesis was increased in these plants, despite the lack of an increase in the JA–isoleucine conjugate level known to regulate the expression of the mentioned genes.…”

Regulation of Sixth Seminal Root Formation by Jasmonate in Triticum aestivum L.

“…The decrease in endogenous jasmonate level resulted from the overexpression of jasmonate biosynthesis pathway genes has been observed in several studies [ 58 , 60 , 61 , 62 ], and the possible mechanisms associated with the negative feedback loops, regulating jasmonate levels, or the inhibition of enzymatic activity of OPR due to the dimerization of proteins have been previously discussed [ 45 , 46 , 47 , 56 , 63 ]. Unexpectedly, the rate of SR-6 formation did not correlate with the level of endogenous jasmonic acid levels ( Figure 4 ).…”

An Optimized Tobacco Hairy Root Induction System for Functional Analysis of Nicotine Biosynthesis-Related Genes

“…Considering that the expression of all the other tested genes in the nicotine synthesis pathway was declined, upregulation of NtMPO2 expression was probably responsible, at least partially, for the increased nicotine accumulation in MsSPL12 transgenic hairy roots. It was previously reported that overexpression of NtAOC2, NtPMT1a, and NtQPT2 did not significantly change the nicotine content [55,56]. Nicotine biosynthesis, therefore, could be synergistically regulated at many levels.…”