molecules Article Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content Ning Yan 1, Hongbo Zhang 1, Zhongfeng Zhang 1, John Shi 2, Michael P. Timko 3, Yongmei Du 1, Xinmin Liu 1 and Yanhua Liu 1,* 1 Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China; [email protected] (N.Y.); [email protected] (H.Z.); [email protected] (Z.Z.); [email protected] (Y.D.); [email protected] (X.L.) 2 Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, ON N1G 5C9, Canada; [email protected] 3 Department of Biology, University of Virginia, Charlottesville, VA 22904, USA; [email protected] * Correspondence: [email protected]; Tel.: +86-532-8870-1035 Academic Editors: Jianbo Xiao, Pinarosa Avato and Baodong Zheng Received: 9 September 2016; Accepted: 11 November 2016; Published: 15 November 2016 Abstract: Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-D-xylulose 5-phosphate synthase (DXS), two 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), two 2-C-methyl-D-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco. Keywords: Nicotiana tabacum; solanesol; RNA-seq; solanesyl diphosphate synthase; gene expression; chlorophyll 1. Introduction Solanesol is a medically important noncyclic terpene alcohol that is synthesized by the condensation of nine isoprene units. The molecule is a precursor of ubiquinone, also known as coenzyme Q10, 0 vitamin K2, and anticancer drugs, including N-solanesyl-N,N -bis(3,4-dimethoxybenzyl) ethylenediamine (SDB) [1–3]. Coenzyme Q10 possesses both antioxidant and antiaging properties and is reported to strengthen the body’s immune system and cardiovascular function, to improve brain health, and to moderate blood lipids. As a result, coenzyme Q10 has potential usefulness in the treatment of migraines, neurodegenerative diseases, hypertension, and cardiovascular diseases [2–5], and it is also being used as a dietary supplement by patients with type-2 diabetes [6]. Meanwhile, vitamin K2 promotes bone formation and mineralization, inhibits bone resorption, prevents and mitigates osteoporosis, promotes Molecules 2016, 21, 1536; doi:10.3390/molecules21111536 www.mdpi.com/journal/molecules Molecules 2016, 21, 1536 2 of 16 Molecules 2016, 21, 1536 2 of 15 blood coagulation, and improves arterial stiffness [7]; and SDB, mediated by P-proteins, is used in theosteoporosis, treatment of promotes several types blood of coagul drugation, resistance and improves in tumours arterial and stiffness plays a synergistic[7]; and SDB, role mediated with certain by antitumorP-proteins, drugs is used [8,9]. in Recently, the treatment Yao et al.of [several10] reported types thatof drug solanesol resistance can protectin tumours human and hepatic plays L02a cellssynergistic from ethanol-induced role with certain oxidative antitumor injury drugs via[8,9]. upregulation Recently, Yao of et HO-1 al. [10] and reported Hsp70 that expression. solanesol can Thus, theprotect medical human benefits hepatic of solanesol L02 cells andfrom its ethanol-induced derivatives are oxidative well established. injury via upregulation of HO-1 and Hsp70The expression. molecule Thus, was firstthe medical isolated benefits from of tobaccosolanesol ( Nicotianaand its derivatives tabacum areL.) well in established. 1956 and has subsequentlyThe molecule been reportedwas first isolated to occur from in tobacco other solanaceous (Nicotiana tabacum plants, L.) includingin 1956 and tomatoes, has subsequently potatoes, eggplants,been reported and peppers to occur [1in,3 ,other11,12 ],solanaceous in which itplan occursts, including in both freetomatoes, and ester-bound potatoes, eggplants, states [3 ,and12,13 ]. peppers [1,3,11,12], in which it occurs in both free and ester-bound states [3,12,13]. Solanesol Solanesol biosynthesis occurs in the plastids of higher plants via the 2-C-methyl-D-erythritol 4-phosphatebiosynthesis (MEP) occurs in pathway the plastids [3, 12of ,higher14], and plants Yan via etthe al.2-C-methyl- [3] andD-erythritol Taylor and 4-phosphate Fraser [12 (MEP)] have pathway [3,12,14], and Yan et al. [3] and Taylor and Fraser [12] have reported that the key enzymes reported that the key enzymes of solanesol biosynthesis include 1-deoxy-D-xylulose 5-phosphate of solanesol biosynthesis include 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D- synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase 4-phosphate cytidylyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4- 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), 1- 4-diphosphatehydroxy-2-methyl-2-( synthaseE)-butenyl (IspG), 1-hydroxy-2-methyl-2-(4-diphosphate reductase E()-butenylIspH), isopentenyl 4-diphosphate diphosphate reductase isomerase (IspH ), isopentenyl(IPI), and diphosphatesolanesyl diphosphate isomerase (IPI),synthase and solanesyl(SPS) (Scheme diphosphate 1) [3,12]. synthase However, (SPS) as (Scheme a long-chain1)[ 3,12 ]. However,polyisoprenoid as a long-chain alcohol, solanesol polyisoprenoid is difficult alcohol, to synthesize solanesol de isnovo difficult [11], and to synthesize solanesol is de primarily novo [11 ], andproduced solanesol via is extraction primarily from produced plants, via particularly extraction tobacco from plants, leaves particularly[3,12]. tobacco leaves [3,12]. SchemeScheme 1. 1.The The solanesol biosynthetic biosynthetic pathway pathway in the in plastids the plastids of tobacco oftobacco plants. Enzymes plants. in Enzymes the solanesol in the biosynthetic pathway are abbreviated as follows: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1- solanesol biosynthetic pathway are abbreviated as follows: DXS, 1-deoxy-D-xylulose 5-phosphate deoxy-D-xylulose 5-phosphate reductoisomerase; IspD, 2-C-methyl-D-erythritol 4-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; IspD, 2-C-methyl-D-erythritol cytidylyltransferase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, 2-C-methyl-D- 4-phosphate cytidylyltransferase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-(E)-butenyl synthase; IspH, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IPI, isopentenyl diphosphate 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IPI, isomerase; SPS, solanesyl diphosphate synthase. isopentenyl diphosphate isomerase; SPS, solanesyl diphosphate synthase. MoleculesMolecules2016 2016, 21, 21, 1536, 1536 3 of3 of15 16 Currently, nothing is known about the expression of genes involved in the biosynthetic pathway Currently, nothing is known about the expression of genes involved in the biosynthetic pathway of solanesol in tobacco, and although some studies have investigated the solanesol content of tobacco of solanesol in tobacco, and although some studies have investigated the solanesol content of tobacco leaves [3,12], little is known about the relative contents of different states of solanesol in different leaves [3,12], little is known about the relative contents of different states of solanesol in different organs organs or at different growing stages. Therefore, in the present study, we investigated the expression orof at solanesol different biosynthesis growing stages. genes Therefore, and the inaccumulation the present study,of solanesol we investigated in three organs the expression (leaves, stems, of solanesol and biosynthesisroots) of tobacco genes plants, and the as well accumulation as in the leaves of solanesol of tobacco in threeplants organs at four (leaves, growing stems, stages. and This roots) work of tobaccoprovides plants, a starting as well point as