Nicotine is one of the most studied secondary metabolites due to the cigarette consumption. Even though it has been used for a long time, how it is regulated in tobacco plant remains unknown. In plant, nicotine functions as a herbivore-preventing agent, growth-regulating factor, and detoxification compound. Nicotine is synthesized in the roots, transported to the shoots, and then stored in leaf vacuoles (Mothes 1954; Dawson and Solt 1959; Saunders 1979). The nicotine biosynthetic pathway was clarified by a sequences of studies, and the pathway is summarized in Figure 1-3 (reviewed by Shoji and Hashimoto 2011).
Many reports indicate that hairy roots massively accumulate corresponding secondary metabolites of the host plant. In 1986, Hamill and coworkers reported that N. rustica hairy root culture induced by A. rhizogenes LBA9402 accumulated 3-fold higher nicotine levels than that of intact roots (Hamill et al. 1986). This result was further proved by Parr and Hamill. They generated hairy roots from different species of Nicotiana, and 4.47- to 58.8-fold increases of nicotine level in hairy roots compared with respective intact roots were observed (Parr and Hamill 1987). In the thesis, we would like to unveil the mechanism underlying huge amounts of secondary metabolites in hairy roots.
Figure 1-3 Biosynthetic pathway, transportation, and storage of nicotine.
Abbreviated proteins: ODC, ornithine decarboxylase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase ; AO, aspartate oxidase; QS, quinolinic acid synthase; QPT, quinolinic acid phosphoribosyl transferase; A622, a p r e s u m a b l e o x i d o r e d u t a s e ; S P D S , s p e r m i d i n e s y n t h a s e ; S A M S , S -adenosylmethionine synthase; and SAMDC, S-adenosylmethione decarboxylase;
MATE1/2, two homologous multidrug and toxic compound extrusion (MATE)-type transporters; JAT1; jasmonate-inducible alkaloid transporter 1. Nicotine was synthesized in root cell, and it would be either sent into root vacuole by MATE1/2-proton anti-porter or transported to leaf through xylem. Both transporters for nicotine xylem loading in root and unloading in leaf are unclear. The nicotine in leaf is sent into vacuole by the MATE like transporter JAT1.
Naturally, nicotine is a defensive toxin against insect herbivores (reviewed by Steppuhn et al. 2004). Jasmonic acid and its derivates (JAs) are closely associated
with defensive responses to stresses, including herbivory and wounding (reviewed by Wasternack 2007; Browse 2009; Wasternack and Hause 2013). Moreover, JAs elicit production of kinds of secondary metabolites (Gundlach et al. 1992). Treating jasmonic acid (JA) or methyl jasmonic acid (meJA) stimulates the nicotine contents in tobacco plants and cell suspension culture by activating genes in nicotine biosynthesis pathway (Imanishi et al. 1998; Shoji et al. 2000). The expression levels of ODC, PMT, MPO, AO, QS, QPT, A622, NtMATE1/2, and NtJAT1, almost all of the known enzymes involved in nicotine biosynthetic pathway, are regulated by the plant hormone JAs (Imanishi et al. 1998; Shoji et al. 2000b; Goossens et al. 2003; Xu and Timko 2004). Moreover, nic2 mutant with low nicotine content showed reduced ethylene responsive factor (ERF) transcripts that are involved in JAs-induced nicotine biosynthesis (Shoji et al. 2010). In the promoter regions of putrescine methyltransferase (PMT) and quinolinate phosphoribosyl transferase (QPT), two key enzymes of nicotine biosynthesis, are found to contain JA-responsive G-box and GGC box motifs (Xu and Timko 2004; De Boer et al. 2011). In the absence of JA, the JA transcriptional repressors JASMONATE ZIM DOMAIN (JAZ) 1-3 might block the PMT transcription by physical interaction with MYB2, which binds to the G-box motif. In the presence of JA, the JAZs were attenuated by proteasome-mediated protein degradation, which resulted in the activation of PMT and QPT to stimulate nicotine biosynthesis (De Boer et al. 2011; Shoji and Hashimoto 2011b; Zhang et al.
2011). In addition, cDNA microarray analysis showed that some APETALA2/
ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes are down-regulated in low nicotine gene mutant nic1/2 (i.e. aabb genotype) (Shoji et al. 2010). In the tobacco hairy root tissues, these AP2/ERF genes are up-regulated after treating with
MeJA, while they are down-regulated after treating with ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Shoji et al. 2010). These AP2/ERF might be the transcriptional activators which bind to GGC box after JA signals (De Boer et al. 2011; Shoji and Hashimoto 2011b).
Treating ethylene decreases the nicotine levels in tobacco. Ethylene and JA interact synergistically or antagonistically in various signals. In the view of nicotine biosynthesis, ethylene down-regulates some structural genes by down-regulating the AP2/ERF (Shoji et al. 2000a; Shoji et al. 2010). Manduca sexta induces the ethylene production to prevent nicotine production (von Dahl et al. 2007). The antagonistic interaction between ethylene and JA responses may ensure the effective nicotine-based defense mechanism. In addition, treating auxin reduces the nicotine levels by an unknown mechanism (Tabata et al. 1971; Takahashi and Yamada 1973). To sum up, nicotine is up-regulated by JA pathway, but it is down-regulated by both ethylene and auxins signals.
There is a controversial idea that tobacco hairy root has higher auxin perceptions and greater nicotine content at the same time. Nicotine regulation in tobacco hairy root is a unique mechanism distinguished from that in normal tissue.
5. Objectives
In plants, secondary metabolites are usually tightly regulated; however, the regulatory rules seem to be broken in hairy root tissues, which leads to massive accumulations. Due to the characteristic, researchers have established hairy root culture to manufacture secondary metabolites for two decades. However, hairy root can only be induced in some dicots and few woody plants, which is a huge limitation in applying hairy root to producing more metabolites from the plant species which are
resistant to A. rhizogenes. To expand the applications, our eventual goals are to find out the molecular mechanisms in rhizogenesis and in metabolite accumulations.
Finally, we hope we can produce plant secondary metabolites either by inducing hairy root formation efficiently or by regulating metabolic flux directly. This will hugely improve the development of pharmacology.
In the fundamental step, we focus on the rol genes, which have been reported to be highly related to rhizogenesis and secondary metabolite accumulations in various plants. We take N. tabacum as a plant model because it has been widely applied in studying hairy root formation. Besides, tobacco contains one of the most studied secondary metabolites, nicotine, and other related alkaloids, which offers a metabolomics model for metabolite accumulations. Via studying the functions of the rol genes in tobacco hairy roots, we may figure out how rol genes alter plant signals and ultimately result in rooting and secondary metabolites accumulating. In the case of rooting, we might generate root systems in pharmaceutically valuable monocots or in other plants whose hairy roots cannot be generated through altering the signals based on our findings. Besides, secondary metabolites are up-regulated by rol genes in many types of plant tissues. The enhancement seems to be a general phenomenon without tissue specificity. By studying how the rol genes regulate metabolites, we might contribute to clarify the metabolomics regulatory mechanisms in plants;
furthermore, we could apply the result to enhancing the metabolite productivity and to lower production cost.
Chapter 2: Materials and Methods
1. General DNA manipulation