Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum

Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf... Abstract Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants. Introduction The common tobacco (Nicotiana tabacum L.) is an allotetraploid species which is generated by interspecific hybridization between the ancestors of N. tomentosiformis and N. sylvestris approximately 200,000 years before present. Glandular trichomes on the surface of tobacco produce diterpenoids and sucrose esters comprising short chain fatty acids, together representing up to 30% of the dry weight of the leaf (Wagner 1991). In these exudates, two types of diterpenes produced by Nicotiana spp, polycyclic labdanoids and macrocyclic cembranoids, account for >50% of secretions (Lin and Wagner 1994, Kennedy et al. 1995). Cembranoids, which are biosynthesized by secretory cells in the apex of glandular trichomes and exuded by the trichomes, are secondary metabolites that are relatively abundant in the Nicotiana and Pinus genera in the form of cembratrien-ols (CBT-ols) and cembratrien-diols (CBT-diols) (El Sayed and Sylvester 2007, Liu et al. 2015). Furthermore, tobacco-derived cembranoids are known to have antibacterial, antitumor, antifungal and neuroprotective properties in humans, and anti-human immunodeficiency virus activity (Zubair et al. 2014, Duan et al. 2015, Martins et al. 2015, Ning et al. 2017). Given these properties, it is likely that future synthetic biology research will be concerned with the metabolic engineering of cembranoids. Cembranoid biosynthesis comprises two enzymatic steps (Fig. 1). The first step begins with the cyclization of geranylgeranyl diphosphate (GGPP), which is catalyzed by cembratrien-ol synthase (CBTS) and produces α- and β-CBT-ols (Guo and Wagner 1995, Yan et al. 2016). In the second step, CBT-ols are hydroxylated into CBT-diols, a process catalyzed by Cyt P450 oxygenase (CYP450) (Wang et al. 2001). Thus, CBTS and CYP450 comprise the committed steps in the production of CBT-ols and CBT-diols, respectively. Such biosynthetic pathways help to elucidate the role of exuded substances in plant defense. Studies concerning CBTS in tobacco have identified one candidate cDNA sequence for CBTS (NtCYC1), the silencing of which leads to reduced CBT-ol and CBT-diol production (Wang and Wagner 2003). Transient expression CBT2a in Nicotiana benthamiana led to an increase in the amount of cembratrien-ol production (Bruckner and Tissier 2013). Promoter analysis indicates that β-glucuronidase (GUS) gene expression driving by the NsCBTS2a promoter was restricted exclusively to glandular trichomes (Ennajdaoui et al. 2010). Similarly, a CYP450-encoding gene (NtCYP71D16) is expressed exclusively in glandular trichomes of tobacco plants (Wang et al. 2002). Thus, plant cembranoid biosynthesis proceeds in the plastids from GGPP, which is expressed in all tissues (Tholl 2006, Cheng et al. 2007), while cembranoid secretions are only produced in gland cells for the exclusive expression of CBTS and CYP450 in glandular trichomes. Furthermore, suppression of NtCYP71D16 results in a raised CBT-ol:CBT-diol ratio, thereby increasing aphid resistance (Wang et al. 2001, Wang et al. 2004). However, it is unknown whether the increased aphid resistance detected in NtCYP71D16-suppressed tobacco plants was caused by an increase in CBT-ol production, or by reduced attraction among CBT-diols. Thus, knowledge concerning the biological function of CBTS is fragmentary, and the specific functions of CBT-ols and CBT-diols in aphid resistance remain enigmatic. Fig. 1 View largeDownload slide Plant isoprenoid biosynthesis proceeds from geranylgeranyl diphosphate (GGPP). Based on the pathway network constructed by Vranova et al. (2011). Fig. 1 View largeDownload slide Plant isoprenoid biosynthesis proceeds from geranylgeranyl diphosphate (GGPP). Based on the pathway network constructed by Vranova et al. (2011). In a previous study, we used comparative analysis of microarray transcriptomes between leaves with trichochomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’ to show that a NtCBTS2 gene is expressed exclusively and abundantly in trichomes (Cui et al. 2011). Building on this previous study, the objectives of the study were (i) to obtain a high yield of cembratrien-ols in exudate of glandular trichomes through overexpressing NtCBTS under the control of a trichome-specific promoter; (ii) to analyze whether CBT-ols could be produced in mesophyll cells using constitutive expression of NtCBTS; and (iii) to examine the biological functions of CBT-ols and CBT-diols in aphid resistance. Results Molecular characterization of CBT-ol synthase In our previous study, comparative analysis of microarray transcriptomes between leaves with trichomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’. In our previous study, comparative analysis of microarray transcriptomes between leaves with trichomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’ indicated that a cembratrien-ol synthase 2 gene (CBTS2) was expressed exclusively and abundantly in trichomes (Cui et al. 2011). The putative full-length NtCBTS2 cDNA was assembled by in silico cloning based on the candidate expressed sequence tag (EST) of NtCBTS2 taken from the trichome cDNA library. Sequence analysis identified two genotypes of NtCBTS2, which we designated NtCBTS2a and NtCBTS2b, in tetraploid tobacco (N. tabacum). The two genotypes were identified in N. sylvestris and N. tabacum, and no transcript of NtCBTS2 was present in N. tomentosiformis, indicating that NtCBTS2a and NtCBTS2b may be derived from the parent diploid species N. sylvestris (Supplementary Fig. S1). These results were verified using transcriptome data from N. tabacum, N. tomentosiformis and N. sylvestris retrieved from the Sol Genomics Network. Phylogenetic analysis based on the putative amino acid sequence of the product of the NtCBTS2 gene suggested that the two genotypes detected in N. tabacum were independently derived from CBTS2 genes in N. sylvestris. Additionally, NtCYC1-1 in N. tabacum T.I.1068 (AY495694) was clustered into the CBTS2a clade. Expression patterns of two NtCBTS2 genotypes in various tobacco tissues Cembranoids, existing as CBT-ols and CBT-diols, are widespread in the exudations on the surface of the leaves and flowers in the Nicotiana genus (Severson et al. 1985). Here, NtCBTS2a and NtCBTS2b had similar expression profiles in different tissues of tobacco (Fig. 2), and were expressed primarily in leaf trichomes. Transcription levels were lower in flowers and stems, were minor in roots and were not detected at all in leaves without trichomes. The similar expression profiles indicated that NtCBTS2 isogenes all originated from N. sylvestris, and may possess a similar function in tobacco. Fig. 2 View largeDownload slide Expression patterns of two genotypes of NtCBTS2 in various tobacco tissues. Expression levels of NtCBTS2 were detected using semi-quantitive RT–PCR (gels) and qRT-PCR (histograms). L25 was used as an internal control. The expression of NtCBTS2 in roots was regarded as standard and other values were compared with it. Values are means ± SD (n = 3). LT, leaf trichome; LWT, leaf without trichome. Fig. 2 View largeDownload slide Expression patterns of two genotypes of NtCBTS2 in various tobacco tissues. Expression levels of NtCBTS2 were detected using semi-quantitive RT–PCR (gels) and qRT-PCR (histograms). L25 was used as an internal control. The expression of NtCBTS2 in roots was regarded as standard and other values were compared with it. Values are means ± SD (n = 3). LT, leaf trichome; LWT, leaf without trichome. Subcellular localization Subcellular localization is a common and efficient method of elucidating the protein function, and demonstrating the cellular localization using green fluorescent protein (GFP) fusion proteins in living cells is a frequently used method for measuring cellular distributions of a protein (Hanson and Köhler 2001). Although cembranoid synthesis is thought to occur in the plastid (Tholl 2006, Cheng et al. 2007), no direct cellular localization assays have verified this claim. NtCBTS2b was predicted using the ProtComp program (http://www.softberry.com/) to have a transit peptide to target the protein to the chloroplast. To detect the subcellular localization of NtCBTS2b in living cells, the fusion protein NtCBTS2b–GFP was transiently expressed in tobacco leaves. As shown in Fig. 3, NtCBTS2b was present in chloroplasts. Fig. 3 View largeDownload slide Subcellular localization of NtCBTS2b. NtCBTS2b–GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in tobacco leaves. 1, GFP fluorescence; 2, Chl autofluorescence; 3, merged images; 4, bright field images. Scale bar = 25 μm. Fig. 3 View largeDownload slide Subcellular localization of NtCBTS2b. NtCBTS2b–GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in tobacco leaves. 1, GFP fluorescence; 2, Chl autofluorescence; 3, merged images; 4, bright field images. Scale bar = 25 μm. Transcription levels and protein abundance in transgenic lines Analysis of transcription levels and protein abundance showed that the expression pattern of NtCBTS2b in N. tabacum was similar to that of the diploid genome donor N. sylvestris, whereas NtCBTS2 was not transcribed and translated in N. tomentosiformis (Fig. 4). The result agreed with the sequence analysis of NtCBTS2. Furthermore. the transcription levels of NtCBTS2b in transgenic lines differed significantly. Transcription and protein levels of NtCBTS2 in the 35S:NtCBTS2b and CYP:NtCBTS2b transgenic lines were higher than those in the control lines, and expression of NtCBTS2 in the three NtCBTS2 Ri lines was greatly suppressed. Fig. 4 View largeDownload slide Identification of transgenic tobacco plants. (a) Expression levels of NtCBTS2b in transgenic tobacco plants and controls. The lowest expression of NtCBTS2b (except N. tomentosiformis) was regarded as a standard. Values are means ± SD (n = 3). (b) Western hybridization of NtCBTS2 protein. The membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). Fig. 4 View largeDownload slide Identification of transgenic tobacco plants. (a) Expression levels of NtCBTS2b in transgenic tobacco plants and controls. The lowest expression of NtCBTS2b (except N. tomentosiformis) was regarded as a standard. Values are means ± SD (n = 3). (b) Western hybridization of NtCBTS2 protein. The membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). Effects of NtCBTS2b on glandular trichome cell morphology Cembranoids are biosynthesized by secretory cells located at the apex of the glandular trichomes. Rhodamine staining patterns showed that overexpression of NtCBTS2b caused larger secretory cells to develop in glandular trichomes, whereas suppressing NtCBTS2 activity resulted in smaller secretory cells (Fig. 5). These data implied that overexpression of NtCBTS2b was beneficial to the growth and development of glandular cells. Fig. 5 View largeDownload slide Trichome morphological observation of transgenic lines. (a) Trichome morphology comparison between transgenic lines and controls. (b) Comparison of the diameter of secretory cells of glandular trichomes between transgenic lines and controls. CK, non-transformed tobacco plants; 35S:NtCBTS2b, constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpression of NtCBTS2b specifically in trichomes;NtCBTS2-RNAi, suppression of the NtCBTS2 gene by RNA interference (RNAi). Values are means ± SE (n = 100). a−dValues followed by different letters are significantly different at P < 0.05. Fig. 5 View largeDownload slide Trichome morphological observation of transgenic lines. (a) Trichome morphology comparison between transgenic lines and controls. (b) Comparison of the diameter of secretory cells of glandular trichomes between transgenic lines and controls. CK, non-transformed tobacco plants; 35S:NtCBTS2b, constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpression of NtCBTS2b specifically in trichomes;NtCBTS2-RNAi, suppression of the NtCBTS2 gene by RNA interference (RNAi). Values are means ± SE (n = 100). a−dValues followed by different letters are significantly different at P < 0.05. Expression of NtCBTS2b in trichomes was necessary for CBT-ol biosynthesis To elucidate the specific function of NtCBTS2b in CBT-ol biosynthesis, levels of Chl, carotenoid, gibberellin, ABA and cembranoid, all of which share the same precursor (GGPP), were measured in 10-week-old control and transgenic lines. It was found that there were no significant differences between NtCBTS2b transgenic tobacco and control plants in terms of Chl, carotenoid, ABA or alkane levels (Supplementary Table S2). However, yields of sugar esters, gibberellins and cembranoids in surface extracts from NtCBTS2b-overexpressing lines were significantly higher than those from the control lines (Fig. 6; Table 1). Furthermore, levels of sugar esters, CBT-ols and CBT-diols were higher in the CYP:NtCBTS2b lines than in the 35S:NtCBTS2b lines. Suppressed NtCBTS2 activity resulted in significantly lower levels of cembranoids. These data indicated that the NtCBTS2b gene played a pivotal role in the CBT-ol biosynthesis, and overexpression of NtCBTS2b in glandular trichomes could efficiently increase the amounts of sugar esters and cembranoids in glandular cells. Table 1 Gibberellins, ABA and leaf exudate analysis   α-CBT-ol (μg cm−2)  β-CBT-ol (μg cm−2)  α-CBT-diol (μg cm−2)  β-CBT-diol (μg cm−2)  CBT-diol isomer (μg cm−2)  Sugar ester (μg cm−2)  Gibberellin (ng g−1)  CK  0.31 ± 0.05d  0.12 ± 0.02d  20.84 ± 1.01c  10.61 ± 0.32c  1.24 ± 0.14b  1.47 ± 0.16b  19.23 ± 0.94b  35S:NtCBTS2b  L1  0.74 ± 0.07c  0.24 ± 0.02c  31.57 ± 1.47b  17.56 ± 0.61b  1.85 ± 0.17a  2.08 ± 0.21a  25.67 ± 1.21a  L3  0.84 ± 0.08c  0.20 ± 0.02c  29.24 ± 1.35b  16.94 ± 0.57b  1.77 ± 0.16a  2.06 ± 0.20a  24.35 ± 1.16a  L4  0.67 ± 0.07c  0.21 ± 0.03c  32.94 ± 1.56b  18.74 ± 0.67b  1.92 ± 0.18a  2.15 ± 0.22a  26.34 ± 1.37a  CYP:NtCBTS2b  L1  2.34 ± 0.22b  0.64 ± 0.07b  44.28 ± 1.87a  27.67 ± 1.12a  1.95 ± 0.18a  2.16 ± 0.21a  24.39 ± 1.18a  L3  2.61 ± 0.27b  0.77 ± 0.08b  42.37 ± 1.71a  27.19 ± 1.10a  1.89 ± 0.18a  2.20 ± 0.24a  26.15 ± 1.34a  L4  2.48 ± 0.23b  0.67 ± 0.07b  46.7 1± 1.92a  28.63 ± 1.18a  2.07 ± 0.19a  2.24 ± 0.25a  26.75 ± 1.38a  NtCBTS2-Ri  L1  0.10 ± 0.02e  0.04 ± 0.01e  9.44 ± 0.60d  0.28 ± 0.03e  0.07 ± 0.01c  0.77 ± 0.10c  13.24 ± 0.53c  L3  0.15 ± 0.02e  0.07 ± 0.01e  12.78 ± 0.77d  0.34 ± 0.05e  0.08 ± 0.01c  0.86 ± 0.11c  12.36 ± 0.44c  L4  0.13 ± 0.02e  0.05 ± 0.01e  10.36 ± 0.71d  0.27 ± 0.03e  0.07 ± 0.01c  1.02 ± 0.13c  14.06 ± 0.62c  NtCYP71D16-Ri  L1  8.27 ± 0.51a  2.04 ± 0.21a  8.36 ± 0.52d  1.35 ± 0.15d  0.09 ± 0.01c  0.81 ± 0.12c  11.74 ± 0.40c  L3  11.09 ± 0.78a  3.17 ± 0.25a  5.97 ± 0.46d  1.17 ± 0.11d  0.08 ± 0.01c  0.74 ± 0.11c  12.14 ± 0.44c  L4  8.51 ± 0.52a  2.15 ± 0.21a  9.85 ± 0.61d  1.64 ± 0.16d  0.07 ± 0.01c  0.71 ± 0.11c  13.72 ± 0.61c    α-CBT-ol (μg cm−2)  β-CBT-ol (μg cm−2)  α-CBT-diol (μg cm−2)  β-CBT-diol (μg cm−2)  CBT-diol isomer (μg cm−2)  Sugar ester (μg cm−2)  Gibberellin (ng g−1)  CK  0.31 ± 0.05d  0.12 ± 0.02d  20.84 ± 1.01c  10.61 ± 0.32c  1.24 ± 0.14b  1.47 ± 0.16b  19.23 ± 0.94b  35S:NtCBTS2b  L1  0.74 ± 0.07c  0.24 ± 0.02c  31.57 ± 1.47b  17.56 ± 0.61b  1.85 ± 0.17a  2.08 ± 0.21a  25.67 ± 1.21a  L3  0.84 ± 0.08c  0.20 ± 0.02c  29.24 ± 1.35b  16.94 ± 0.57b  1.77 ± 0.16a  2.06 ± 0.20a  24.35 ± 1.16a  L4  0.67 ± 0.07c  0.21 ± 0.03c  32.94 ± 1.56b  18.74 ± 0.67b  1.92 ± 0.18a  2.15 ± 0.22a  26.34 ± 1.37a  CYP:NtCBTS2b  L1  2.34 ± 0.22b  0.64 ± 0.07b  44.28 ± 1.87a  27.67 ± 1.12a  1.95 ± 0.18a  2.16 ± 0.21a  24.39 ± 1.18a  L3  2.61 ± 0.27b  0.77 ± 0.08b  42.37 ± 1.71a  27.19 ± 1.10a  1.89 ± 0.18a  2.20 ± 0.24a  26.15 ± 1.34a  L4  2.48 ± 0.23b  0.67 ± 0.07b  46.7 1± 1.92a  28.63 ± 1.18a  2.07 ± 0.19a  2.24 ± 0.25a  26.75 ± 1.38a  NtCBTS2-Ri  L1  0.10 ± 0.02e  0.04 ± 0.01e  9.44 ± 0.60d  0.28 ± 0.03e  0.07 ± 0.01c  0.77 ± 0.10c  13.24 ± 0.53c  L3  0.15 ± 0.02e  0.07 ± 0.01e  12.78 ± 0.77d  0.34 ± 0.05e  0.08 ± 0.01c  0.86 ± 0.11c  12.36 ± 0.44c  L4  0.13 ± 0.02e  0.05 ± 0.01e  10.36 ± 0.71d  0.27 ± 0.03e  0.07 ± 0.01c  1.02 ± 0.13c  14.06 ± 0.62c  NtCYP71D16-Ri  L1  8.27 ± 0.51a  2.04 ± 0.21a  8.36 ± 0.52d  1.35 ± 0.15d  0.09 ± 0.01c  0.81 ± 0.12c  11.74 ± 0.40c  L3  11.09 ± 0.78a  3.17 ± 0.25a  5.97 ± 0.46d  1.17 ± 0.11d  0.08 ± 0.01c  0.74 ± 0.11c  12.14 ± 0.44c  L4  8.51 ± 0.52a  2.15 ± 0.21a  9.85 ± 0.61d  1.64 ± 0.16d  0.07 ± 0.01c  0.71 ± 0.11c  13.72 ± 0.61c  CK, non-transformed tobacco plants; 35S:NtCBTS2b. constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpressing NtCBTS2b specifically in trichomes; NtCBTS2-Ri, suppression of the NtCBTS2 gene by RNAi; NtCYP71D16-Ri, suppression of the NtCYP71D16 gene by RNAi. Values are means ± SE (n = 50). a–eValues followed by different letters were significantly different at P <0.05. Fig. 6 View largeDownload slide Gas chromatographic analyses of the cembranoid contents in transgenic tobacco plants. Fifty leaf discs were punched from leaves, and analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. Fig. 6 View largeDownload slide Gas chromatographic analyses of the cembranoid contents in transgenic tobacco plants. Fifty leaf discs were punched from leaves, and analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. Constitutive expression of NtCBTS2b led to cembratrien-ol production in leaf mesophylls The precursor of cembranoid biosynthesis (GGPP) can be synthesized in all plant tissues, while cembranoid secretions are only produced in gland cells, and not detected in leaf mesophylls. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls using bioreactor engineering, the mesophylls of 35S:NtCBTS2b transgenic tobacco were used to analyze cembranoid contents. As predicted, CBT-ols and CBT-diols did not exist in the mesophylls of non-transformed tobacco plants (controls), while lots of CBT-ols and no CBT-diols were observed in the mesophylls of 35S:NtCBTS2b tobacco (Fig. 7). Moreover, there was no significant difference between 35S:NtCBTS2b tobacco and controls in terms of Chls, carotenoids, ABA and gibberellins (data not shown). Therefore, constitutive expression of NtCBTS2b may be a practical method to harvest large amounts of cembratrien-ols using genetic engineering. Fig. 7 View largeDownload slide Production of CBT-ols in mesophylls. (a) Gas chromatographic analyses of the cembranoid contents in the mesophylls of 35S:NtCBTS2b tobacco plants. Fifty leaf discs were punched from leaves and dipped in methylene chloride eight times to remove leaf exudates. The residual leaves without leaf exudates were analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. (b) Chart of the CBT-ols in the mesophylls of 35S:NtCBTS2b tobacco plants quantified by GC-MS. Fig. 7 View largeDownload slide Production of CBT-ols in mesophylls. (a) Gas chromatographic analyses of the cembranoid contents in the mesophylls of 35S:NtCBTS2b tobacco plants. Fifty leaf discs were punched from leaves and dipped in methylene chloride eight times to remove leaf exudates. The residual leaves without leaf exudates were analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. (b) Chart of the CBT-ols in the mesophylls of 35S:NtCBTS2b tobacco plants quantified by GC-MS. Aphid infestation In previous studies, a CYP450-encoding gene (NtCYP71D16) specific to the trichome gland was identified, and the suppression of NtCYP71D16 led to a raised CBT-ol:CBT-diol ratio, which in turn promoted aphidicidal activity (Wang et al. 2001, Wang et al. 2004). To determine whether the promoted aphidicidal activity was due to an increase in CBT-ol production or the reduced attraction of CBT-diols, transgenic N. tabacum lines with different levels of CBT-ols or CBT-diols, labeled 35S:NtCBTS2b, NtCBTS2-Ri, CYP:NtCBTS2b and NtCYP71D16-Ri, were created using methods described previously (Wang et al. 2001) (Supplementary Fig. S2). In the bioactivity assay, the trichome exudates of wild-type and transgenic lines were individually applied on the leaf discs of wild-type tobacco (Fig. 8). Compared with controls, the number of aphids was more on the leaf discs of NtCBTS-Ri lines, fewer on 35S:NtCBTS2b lines and marginal on CYP:NtCBTS2b and NtCYP71D16-Ri lines. It was suggested that accumulation of CBT-ols may help to reduce aphid attraction. In accordance with the bioactivity assay, these transgenic lines had different levels of aphid resistance. As shown in Fig. 9, compared with controls, CYP:NtCBTS2b lines had enhanced CBT-ol and CBT-diol levels, while NtCYP71D16-Ri lines possessed increased CBT-ols and decreased CBT-diol levels. Notably, CYP:NtCBTS2b and NtCYP71D16-Ri lines were equally resistant to aphid colonization, and were more resistant than control lines. Resistance in the 35S:NtCBTS2b lines was also higher than in control lines, whereas it was lower than in CYP:NtCBTS2b and NtCYP71D16-Ri lines. It was noteworthy that the heaviest infestation was observed in NtCBTS-Ri lines, which had the lowest CBT-ol levels. These results indicated that accumulation of CBT-ols helped to elevate the aphid resistance of plants, and the level of CBT-diol was not related to the aphidicidal activity. Fig. 8 View largeDownload slide Bioactivity assays of the trichome exudates of wild-type and transgenic lines. (a) Colonization responses of aphids on the trichome exudates of wild-type and transgenic lines. (b) Aphid numbers on the wild-type leaf discs with different trichome exudates. Values are means ± SE (n = 10). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 8 View largeDownload slide Bioactivity assays of the trichome exudates of wild-type and transgenic lines. (a) Colonization responses of aphids on the trichome exudates of wild-type and transgenic lines. (b) Aphid numbers on the wild-type leaf discs with different trichome exudates. Values are means ± SE (n = 10). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 9 View largeDownload slide Aphid infestation response on different levels of CBT-ols. (a) Colonization responses of aphids on NtCBTS2b transgenic lines, NtCYP71D16-Ri lines and controls. (b) Survival of aphids on transgenic lines and controls. Values are means ± SE (n = 20). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 9 View largeDownload slide Aphid infestation response on different levels of CBT-ols. (a) Colonization responses of aphids on NtCBTS2b transgenic lines, NtCYP71D16-Ri lines and controls. (b) Survival of aphids on transgenic lines and controls. Values are means ± SE (n = 20). a–dValues followed by different letters are significantly different at P < 0.05. Discussion The results of our study clarify our objectives, indicating that (i) NtCBTS2b plays a pivotal role in CBT-ol biosynthesis; NtCBTS2b overexpression promotes sugar ester and cembranoid production in glandular trichome cells; and accumulating CBT-ols promote aphid resistance; (ii) overexpression of NtCBTS2b may substantially promote the growth and development of glandular cells; and (iii) constitutive expression of NtCBTS2b led to cembratrien-ol production also in mesophylls, not only in glandular trichomes. Our data also indicate that NtCBTS is not transcribed and translated in N. tomentosiformis. Finally, our results confirm that NtCBTS2b is distributed in chloroplasts, confirming a notion long believed that, to date, had not yet been verified. Together, these data provide crucial insights into the biological function of NtCBTS2b in N. tabacum. As an important economic crop with large yields, tobacco leaves are harvested for a wide range of applications in the medical, food and biofuel industries. Tobacco-derived cembranoids are known to show antifungal, antibacterial, anti-human immunodeficiency virus, antitumor and neuroprotective properties (Zubair et al. 2014, Duan et al. 2015, Martins et al. 2015, Ning et al. 2017), while cembranoid secretions are only produced in glandular trichomes, and thereby the quantity produced is limited. Considering that the precursor of cembranoid biosynthesis (GGPP) can be synthesized in all plant tissues, it was possible to engineer a platform for cembratrien-ol production in leaf mesophylls through expressing NtCBTS2 in leaf mesophylls. Our results suggested that it was feasible to produce natural product cembratrien-ols in tobacco mesophylls using constitutive expression of NtCBTS2b. In future cembratrien-diol synthetic biology studies, the focus could be on the simultaneously constitutive expression of NtCBTS2b and NtCYP71D16 using genetic engineering. Our results demonstrate that overexpression or knockdown of NtCBTS2b did not suppress growth and development in N. tabacum. Indeed, the overexpression of NtCBTS2b substantially promotes the growth and development of glandular cells. One explanation for this increase in growth and development may be that the gibberellin concentration increased in NtCBTS2b-overexpressing lines. This result suggests that NtCBTS2b may be overexpressed in commercial plants with few or no adverse effects on plant growth, and that such overexpression promotes the production of such economically important compounds as cembranoids. Additionally, accumulation of CBT-ols promotes aphid resistance. Overexpressing NtCBTS2b specifically in glandular trichomes had similar aphidicidal activity to suppressing NtCYP71D16, even though the level of CBT-ols in NtCYP71D16-Ri lines was higher than that in CYP:NtCBTS2b lines. Noticeably, NtCYP71D16-suppressed transgenic lines produced less of the primary trichome exudations (CBT-diol), and produced more of its precursor (CBT-ol) instead. Previous studies have reported that the CBT-diols produced by tobacco plants inhibit growth in Valsa mali (Duan et al. 2015, Yan et al. 2017). Thus, overexpressing NtCBTS2b specifically in glandular trichomes may be an ideal method for promoting aphid resistance in tobacco taxa. Our study primarily focused on morphological traits, physiological parameters and aphid resistance in NtCBTS2b transgenic N. tabacum lines. We conclude that NtCBTS2b plays a key role in CBT-ol biosynthesis; CBT-ols plays important roles in aphid resistance; constitutive expression of NtCBTS2b leads to cembratrien-ol production in mesophylls rather than only in glandular trichomes; and overexpressing NtCBTS2b specifically in glandular trichomes is greatly important for promoting the aphid resistance of plants. Materials and Methods Plant materials Nicotiana tabacum ‘Kentucky 326’ plants were cultured in a greenhouse with a light/dark cycle of 14/10 h at 30/24°C. At the flowering stage, glandular trichomes, leaves with trichomes, leaves lacking trichomes, flowers, stems and roots were excised. Leaf trichomes were obtained by freezing leaves (length: 15 cm) on an aluminum sheet over a liquid nitrogen bath. Once frozen, a paintbrush was used to collect trichomes by brushing, and remaining leaf tissues were sampled as leaves lacking trichomes. Total RNA was isolated using TRIzol reagent (TAKARA), and then treated with RNase-free DNase I (TAKARA). Sequence analysis of full-length NtCBTS cDNA To obtain the coding region sequence of NtCBTS2, two primers were designed based on the lateral flanking sequences of the coding region. NCBI (https://www.ncbi.nlm.nih.gov/) and SGN (http://solgenomics.net/) databases were searched using BLAST to identify matches between deduced nucleotide and amino acid sequences and registered sequences. Sequence alignment was carried out using megAlign in DNAStar. The PROSITE database (http://expasy.hcuge.ch/sprot/prosite.html) was used to identify possible activity sites and functional regions. A total of 18 primers were used throughout all genetic analyses (Supplementary Table S1). NtCBTS2 expression patterns Semi-quantitative reverse transcription–PCR (RT–PCR) analysis and quantitative real-time PCR (qRT-PCR) were performed to detect the mRNA transcription levels of NtCBTS2 in different tobacco tissues. Expression of the ribosomal protein gene L25 (Volkov et al. 2003) was used as internal standard. Specific primers were designed based on sequence variations in NtCBTS2a and NtCBTS2b (Supplementary Table S1). qRT-PCR was performed to detect NtCBTS2 transcription levels in transgenic tobacco tissues. qRT-PCR was carried out with an ABI PRISM 7000 system (Applied Biosystems). Transcription levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001). Subcellular localization The coding sequence of NtCBTS2b was inserted upstream of GFP and downstream of 35S in the pJIT163-GFP vector for constructing an NtCBTS2b–GFP fusion protein. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Positive clones were infiltrated into tobacco leaves with a 1 ml syringe. Epidermal cells of infiltrated leaves were observed for green fluorescence with a laser-scanning confocal microscope (Leika TCS-NT; Leika) following 48 h incubation. Generation of transgenic plants Reverse and forward genetic approaches are commonly used to discern pivotal molecular factors. To determine the precise role of CBT-ols in N. tabacum, transgenic lines were generated in which NtCBTS2b was either overexpressed or silenced by RNA interference. Phenotypes of these different transgenic lines were characterized at different developmental stages. To test the effects of NtCBTS2b overexpression, the coding region of NtCBTS2b was separately inserted downstream of the constitutive CaMV 35S promoter in a pCAMBIA 1301 plasmid (Cambia), and downstream of a NtCYP450 trichome-specific promoter (Wang et al. 2002) in a modified pCAMBIA 1391 plasmid, yielding the p35S:NtCBTS2b and pCYP:NtCBT2Sb constructs, respectively. To test the effects of NtCBTS2 suppression, a 287 bp fragment of the NtCBTS2 coding sequence was ligated into the pK7GWIWG2(II) expression vector (Karimi et al. 2002). The recombinant vectors were separately transformed into tobacco using A. tumefaciens strain EHA105. Gene expression in NtCBTS2b transgenic lines For each construct, 15 independent transgenic lines were generated. T3 homozygous lines were selected based on hygromycin segregation in T1 and T2 seeds tested using χ2 tests for further analysis. The successful integration of recombinant vector into the genomes of transgenic lines was determined using DNA-PCR (Supplementary Fig. S3). To detect the effects of NtCBTS2b overexpression and knockdown in transgenic plants, four transgenic lines were used for the measurement of gene expression and protein abundance levels. Three control lines, i.e. N. tabacum ‘Kentucky 326’ and its diploid genome donors (N. tomentosiformis and N. sylvestris), were also used in qPCR and Western blotting analyses. NtCBTS protein level assays Proteins were isolated and quantified from approximately 0.1 g of four-leaf-old tobacco seedlings as described previously (Bradford 1976). A 20 μg aliquot of protein from each sample was electrophoretically fractioned in a 12.5% polyacrylamide gel and transferred onto a nitrocellulose membrane. Membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). The membrane was exposed to X-ray film (Fujifilm) to detect signals. Trichome morphology observations Seedlings were stained using 2% rhodamine at 37°C for 2 h, and then rinsed with ddH2O. Thirty seedling leaves of identical age and position were detached from transgenic plants (3 weeks old). Trichomes were photographed with an Axioplan 2 microscope (Carl Zeiss AG). Leaf trichom density, the area of glandular cells and the length and width of each trichome were recorded. Analysis of leaf surface exudates and isoprenoid contents Isoprenoid contents in transgenic lines were determined using methods described previously (Zhang et al. 2015). Gibberellin, ABA, IAA and zeatin riboside contents were measured using enzyme-linked immunosorbent assays (ELISAs). To isolate diterpenoid compounds produced in leaves, 50 leaf discs (diameter: 2 cm) were excised from leaves (leaf width: 10 cm) and immersed in 600 ml of methylene chloride for 2 s eight times. Leaf surface exudates were analyzed using gas chromatography–mass spectrometry (GC-MS). To detect whether diterpenoids are produced in the mesophylls of 35S:NtCBTS2b transgenic tobacco plants, these residual leaves after removing leaf exudates were pre-frozen in liquid nitrogen, and ground to a powder to analyze CBT-ols and CBT-diols. To measure carotenoid and Chl contents, 0.1 g of trichomes or 0.2 g of leaves were powdered and extracted with 25 ml of 90% acetone using ultrasonic extraction under shading conditions. Supernatants were collected and filtered through a 0.45 μm PTFE membrane filter. Pigment extracts were determined using reverse-phase HPLC. Bioactivity assays Fifty leaf discs (diameter: 2 cm) of wild-type or transgenic lines (35S:NtCBTS2b, CYP:NtCBTS2b, NtCBTS2-Ri and NtCYP71D16-Ri) were excised from leaves (leaf width: 10 cm) to collect trichome exudates as described previously (Zhang et al. 2015). Wild-type tobacco plants were cultured for 4 weeks, and leaf discs (diameter: 5 cm) were excised from leaves (leaf width: 10 cm). Ten leaf discs were immersed in the trichome exudate solution extracted from each transgenic line for 5 s, and placed in a closed container at room temperature. Leaf discs were arranged evenly in a circle. Two hundred aphids were released in the center of the circle, and infested these leaf discs naturally. The number of aphids on each leaf disc was counted 2 d later. Bioactivity assays were carried out in triplicate. Aphid infestation The tobacco aphid (Myzus nicotianae), a major tobacco pest with a cosmopolitan distribution, was used in the assay. Transgenic seeds were germinated and cultured in a greenhouse with a light/dark cycle of 14/10 h at 30/24°C. Three weeks later, 1,000 aphids were released and were allowed to infest the plants naturally. The number of aphids infesting each transgenic line was scored after 2 weeks. Aphid infestation experiments were carried out in triplicate. Data analysis All statistical procedures were conducted using the Student’s t-test in SAS v9.2 to detect significant differences between means. The significance threshold was set at 0.05 or 0.01. Supplementary Data Supplementary data are available at PCP online. Funding The work was supported by the State Tobacco Monopoly Administration of China [grant No. 110201401003 (JY-03)]. Disclosures The authors have no conflicts of interest to declare. References Bradford M.M. ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem.  72: 248– 254. Google Scholar CrossRef Search ADS PubMed  Bruckner K., Tissier A. ( 2013) High-level diterpene production by transient expression in Nicotiana benthamiana. Plant Methods  9: 46. Google Scholar CrossRef Search ADS PubMed  Cheng A.X., Lou Y.G., Mao Y.B., Lu S., Wang L.J., Chen X.Y. 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Abbreviations Abbreviations CBT-diol cembratrienediol CBT-ol cembratrieneol CBTS cembratrien-ol synthase CYP promoter CYP71D16 trichome-specific promoter CYP450 Cyt P450 oxygenase CYP:NtCBTS2b transgenic plants expressing NtCBTS2b under the control of the CYP promoter GC-MS gas chromatography–mass spectrometry GFP green fluorescent protein GGPP geranylgeranyl diphosphate qRT-PCR quantitative real-time PCR RNAi RNA interference RT–PCR reverse transcription–PCR 35S Cauliflower mosaic virus (CaMV) 35S promoter 35S:NtCBTS2b transgenic plants expressing NtCBTS2b under the control of the 35S promoter NtCBTS2-Ri transgenic plants with NtCBTS2 suppressed by RNA interference © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0032-0781
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10.1093/pcp/pcy004
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Abstract

Abstract Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants. Introduction The common tobacco (Nicotiana tabacum L.) is an allotetraploid species which is generated by interspecific hybridization between the ancestors of N. tomentosiformis and N. sylvestris approximately 200,000 years before present. Glandular trichomes on the surface of tobacco produce diterpenoids and sucrose esters comprising short chain fatty acids, together representing up to 30% of the dry weight of the leaf (Wagner 1991). In these exudates, two types of diterpenes produced by Nicotiana spp, polycyclic labdanoids and macrocyclic cembranoids, account for >50% of secretions (Lin and Wagner 1994, Kennedy et al. 1995). Cembranoids, which are biosynthesized by secretory cells in the apex of glandular trichomes and exuded by the trichomes, are secondary metabolites that are relatively abundant in the Nicotiana and Pinus genera in the form of cembratrien-ols (CBT-ols) and cembratrien-diols (CBT-diols) (El Sayed and Sylvester 2007, Liu et al. 2015). Furthermore, tobacco-derived cembranoids are known to have antibacterial, antitumor, antifungal and neuroprotective properties in humans, and anti-human immunodeficiency virus activity (Zubair et al. 2014, Duan et al. 2015, Martins et al. 2015, Ning et al. 2017). Given these properties, it is likely that future synthetic biology research will be concerned with the metabolic engineering of cembranoids. Cembranoid biosynthesis comprises two enzymatic steps (Fig. 1). The first step begins with the cyclization of geranylgeranyl diphosphate (GGPP), which is catalyzed by cembratrien-ol synthase (CBTS) and produces α- and β-CBT-ols (Guo and Wagner 1995, Yan et al. 2016). In the second step, CBT-ols are hydroxylated into CBT-diols, a process catalyzed by Cyt P450 oxygenase (CYP450) (Wang et al. 2001). Thus, CBTS and CYP450 comprise the committed steps in the production of CBT-ols and CBT-diols, respectively. Such biosynthetic pathways help to elucidate the role of exuded substances in plant defense. Studies concerning CBTS in tobacco have identified one candidate cDNA sequence for CBTS (NtCYC1), the silencing of which leads to reduced CBT-ol and CBT-diol production (Wang and Wagner 2003). Transient expression CBT2a in Nicotiana benthamiana led to an increase in the amount of cembratrien-ol production (Bruckner and Tissier 2013). Promoter analysis indicates that β-glucuronidase (GUS) gene expression driving by the NsCBTS2a promoter was restricted exclusively to glandular trichomes (Ennajdaoui et al. 2010). Similarly, a CYP450-encoding gene (NtCYP71D16) is expressed exclusively in glandular trichomes of tobacco plants (Wang et al. 2002). Thus, plant cembranoid biosynthesis proceeds in the plastids from GGPP, which is expressed in all tissues (Tholl 2006, Cheng et al. 2007), while cembranoid secretions are only produced in gland cells for the exclusive expression of CBTS and CYP450 in glandular trichomes. Furthermore, suppression of NtCYP71D16 results in a raised CBT-ol:CBT-diol ratio, thereby increasing aphid resistance (Wang et al. 2001, Wang et al. 2004). However, it is unknown whether the increased aphid resistance detected in NtCYP71D16-suppressed tobacco plants was caused by an increase in CBT-ol production, or by reduced attraction among CBT-diols. Thus, knowledge concerning the biological function of CBTS is fragmentary, and the specific functions of CBT-ols and CBT-diols in aphid resistance remain enigmatic. Fig. 1 View largeDownload slide Plant isoprenoid biosynthesis proceeds from geranylgeranyl diphosphate (GGPP). Based on the pathway network constructed by Vranova et al. (2011). Fig. 1 View largeDownload slide Plant isoprenoid biosynthesis proceeds from geranylgeranyl diphosphate (GGPP). Based on the pathway network constructed by Vranova et al. (2011). In a previous study, we used comparative analysis of microarray transcriptomes between leaves with trichochomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’ to show that a NtCBTS2 gene is expressed exclusively and abundantly in trichomes (Cui et al. 2011). Building on this previous study, the objectives of the study were (i) to obtain a high yield of cembratrien-ols in exudate of glandular trichomes through overexpressing NtCBTS under the control of a trichome-specific promoter; (ii) to analyze whether CBT-ols could be produced in mesophyll cells using constitutive expression of NtCBTS; and (iii) to examine the biological functions of CBT-ols and CBT-diols in aphid resistance. Results Molecular characterization of CBT-ol synthase In our previous study, comparative analysis of microarray transcriptomes between leaves with trichomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’. In our previous study, comparative analysis of microarray transcriptomes between leaves with trichomes and leaves without trichomes from N. tabacum L. ‘Kentucky 326’ indicated that a cembratrien-ol synthase 2 gene (CBTS2) was expressed exclusively and abundantly in trichomes (Cui et al. 2011). The putative full-length NtCBTS2 cDNA was assembled by in silico cloning based on the candidate expressed sequence tag (EST) of NtCBTS2 taken from the trichome cDNA library. Sequence analysis identified two genotypes of NtCBTS2, which we designated NtCBTS2a and NtCBTS2b, in tetraploid tobacco (N. tabacum). The two genotypes were identified in N. sylvestris and N. tabacum, and no transcript of NtCBTS2 was present in N. tomentosiformis, indicating that NtCBTS2a and NtCBTS2b may be derived from the parent diploid species N. sylvestris (Supplementary Fig. S1). These results were verified using transcriptome data from N. tabacum, N. tomentosiformis and N. sylvestris retrieved from the Sol Genomics Network. Phylogenetic analysis based on the putative amino acid sequence of the product of the NtCBTS2 gene suggested that the two genotypes detected in N. tabacum were independently derived from CBTS2 genes in N. sylvestris. Additionally, NtCYC1-1 in N. tabacum T.I.1068 (AY495694) was clustered into the CBTS2a clade. Expression patterns of two NtCBTS2 genotypes in various tobacco tissues Cembranoids, existing as CBT-ols and CBT-diols, are widespread in the exudations on the surface of the leaves and flowers in the Nicotiana genus (Severson et al. 1985). Here, NtCBTS2a and NtCBTS2b had similar expression profiles in different tissues of tobacco (Fig. 2), and were expressed primarily in leaf trichomes. Transcription levels were lower in flowers and stems, were minor in roots and were not detected at all in leaves without trichomes. The similar expression profiles indicated that NtCBTS2 isogenes all originated from N. sylvestris, and may possess a similar function in tobacco. Fig. 2 View largeDownload slide Expression patterns of two genotypes of NtCBTS2 in various tobacco tissues. Expression levels of NtCBTS2 were detected using semi-quantitive RT–PCR (gels) and qRT-PCR (histograms). L25 was used as an internal control. The expression of NtCBTS2 in roots was regarded as standard and other values were compared with it. Values are means ± SD (n = 3). LT, leaf trichome; LWT, leaf without trichome. Fig. 2 View largeDownload slide Expression patterns of two genotypes of NtCBTS2 in various tobacco tissues. Expression levels of NtCBTS2 were detected using semi-quantitive RT–PCR (gels) and qRT-PCR (histograms). L25 was used as an internal control. The expression of NtCBTS2 in roots was regarded as standard and other values were compared with it. Values are means ± SD (n = 3). LT, leaf trichome; LWT, leaf without trichome. Subcellular localization Subcellular localization is a common and efficient method of elucidating the protein function, and demonstrating the cellular localization using green fluorescent protein (GFP) fusion proteins in living cells is a frequently used method for measuring cellular distributions of a protein (Hanson and Köhler 2001). Although cembranoid synthesis is thought to occur in the plastid (Tholl 2006, Cheng et al. 2007), no direct cellular localization assays have verified this claim. NtCBTS2b was predicted using the ProtComp program (http://www.softberry.com/) to have a transit peptide to target the protein to the chloroplast. To detect the subcellular localization of NtCBTS2b in living cells, the fusion protein NtCBTS2b–GFP was transiently expressed in tobacco leaves. As shown in Fig. 3, NtCBTS2b was present in chloroplasts. Fig. 3 View largeDownload slide Subcellular localization of NtCBTS2b. NtCBTS2b–GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in tobacco leaves. 1, GFP fluorescence; 2, Chl autofluorescence; 3, merged images; 4, bright field images. Scale bar = 25 μm. Fig. 3 View largeDownload slide Subcellular localization of NtCBTS2b. NtCBTS2b–GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in tobacco leaves. 1, GFP fluorescence; 2, Chl autofluorescence; 3, merged images; 4, bright field images. Scale bar = 25 μm. Transcription levels and protein abundance in transgenic lines Analysis of transcription levels and protein abundance showed that the expression pattern of NtCBTS2b in N. tabacum was similar to that of the diploid genome donor N. sylvestris, whereas NtCBTS2 was not transcribed and translated in N. tomentosiformis (Fig. 4). The result agreed with the sequence analysis of NtCBTS2. Furthermore. the transcription levels of NtCBTS2b in transgenic lines differed significantly. Transcription and protein levels of NtCBTS2 in the 35S:NtCBTS2b and CYP:NtCBTS2b transgenic lines were higher than those in the control lines, and expression of NtCBTS2 in the three NtCBTS2 Ri lines was greatly suppressed. Fig. 4 View largeDownload slide Identification of transgenic tobacco plants. (a) Expression levels of NtCBTS2b in transgenic tobacco plants and controls. The lowest expression of NtCBTS2b (except N. tomentosiformis) was regarded as a standard. Values are means ± SD (n = 3). (b) Western hybridization of NtCBTS2 protein. The membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). Fig. 4 View largeDownload slide Identification of transgenic tobacco plants. (a) Expression levels of NtCBTS2b in transgenic tobacco plants and controls. The lowest expression of NtCBTS2b (except N. tomentosiformis) was regarded as a standard. Values are means ± SD (n = 3). (b) Western hybridization of NtCBTS2 protein. The membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). Effects of NtCBTS2b on glandular trichome cell morphology Cembranoids are biosynthesized by secretory cells located at the apex of the glandular trichomes. Rhodamine staining patterns showed that overexpression of NtCBTS2b caused larger secretory cells to develop in glandular trichomes, whereas suppressing NtCBTS2 activity resulted in smaller secretory cells (Fig. 5). These data implied that overexpression of NtCBTS2b was beneficial to the growth and development of glandular cells. Fig. 5 View largeDownload slide Trichome morphological observation of transgenic lines. (a) Trichome morphology comparison between transgenic lines and controls. (b) Comparison of the diameter of secretory cells of glandular trichomes between transgenic lines and controls. CK, non-transformed tobacco plants; 35S:NtCBTS2b, constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpression of NtCBTS2b specifically in trichomes;NtCBTS2-RNAi, suppression of the NtCBTS2 gene by RNA interference (RNAi). Values are means ± SE (n = 100). a−dValues followed by different letters are significantly different at P < 0.05. Fig. 5 View largeDownload slide Trichome morphological observation of transgenic lines. (a) Trichome morphology comparison between transgenic lines and controls. (b) Comparison of the diameter of secretory cells of glandular trichomes between transgenic lines and controls. CK, non-transformed tobacco plants; 35S:NtCBTS2b, constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpression of NtCBTS2b specifically in trichomes;NtCBTS2-RNAi, suppression of the NtCBTS2 gene by RNA interference (RNAi). Values are means ± SE (n = 100). a−dValues followed by different letters are significantly different at P < 0.05. Expression of NtCBTS2b in trichomes was necessary for CBT-ol biosynthesis To elucidate the specific function of NtCBTS2b in CBT-ol biosynthesis, levels of Chl, carotenoid, gibberellin, ABA and cembranoid, all of which share the same precursor (GGPP), were measured in 10-week-old control and transgenic lines. It was found that there were no significant differences between NtCBTS2b transgenic tobacco and control plants in terms of Chl, carotenoid, ABA or alkane levels (Supplementary Table S2). However, yields of sugar esters, gibberellins and cembranoids in surface extracts from NtCBTS2b-overexpressing lines were significantly higher than those from the control lines (Fig. 6; Table 1). Furthermore, levels of sugar esters, CBT-ols and CBT-diols were higher in the CYP:NtCBTS2b lines than in the 35S:NtCBTS2b lines. Suppressed NtCBTS2 activity resulted in significantly lower levels of cembranoids. These data indicated that the NtCBTS2b gene played a pivotal role in the CBT-ol biosynthesis, and overexpression of NtCBTS2b in glandular trichomes could efficiently increase the amounts of sugar esters and cembranoids in glandular cells. Table 1 Gibberellins, ABA and leaf exudate analysis   α-CBT-ol (μg cm−2)  β-CBT-ol (μg cm−2)  α-CBT-diol (μg cm−2)  β-CBT-diol (μg cm−2)  CBT-diol isomer (μg cm−2)  Sugar ester (μg cm−2)  Gibberellin (ng g−1)  CK  0.31 ± 0.05d  0.12 ± 0.02d  20.84 ± 1.01c  10.61 ± 0.32c  1.24 ± 0.14b  1.47 ± 0.16b  19.23 ± 0.94b  35S:NtCBTS2b  L1  0.74 ± 0.07c  0.24 ± 0.02c  31.57 ± 1.47b  17.56 ± 0.61b  1.85 ± 0.17a  2.08 ± 0.21a  25.67 ± 1.21a  L3  0.84 ± 0.08c  0.20 ± 0.02c  29.24 ± 1.35b  16.94 ± 0.57b  1.77 ± 0.16a  2.06 ± 0.20a  24.35 ± 1.16a  L4  0.67 ± 0.07c  0.21 ± 0.03c  32.94 ± 1.56b  18.74 ± 0.67b  1.92 ± 0.18a  2.15 ± 0.22a  26.34 ± 1.37a  CYP:NtCBTS2b  L1  2.34 ± 0.22b  0.64 ± 0.07b  44.28 ± 1.87a  27.67 ± 1.12a  1.95 ± 0.18a  2.16 ± 0.21a  24.39 ± 1.18a  L3  2.61 ± 0.27b  0.77 ± 0.08b  42.37 ± 1.71a  27.19 ± 1.10a  1.89 ± 0.18a  2.20 ± 0.24a  26.15 ± 1.34a  L4  2.48 ± 0.23b  0.67 ± 0.07b  46.7 1± 1.92a  28.63 ± 1.18a  2.07 ± 0.19a  2.24 ± 0.25a  26.75 ± 1.38a  NtCBTS2-Ri  L1  0.10 ± 0.02e  0.04 ± 0.01e  9.44 ± 0.60d  0.28 ± 0.03e  0.07 ± 0.01c  0.77 ± 0.10c  13.24 ± 0.53c  L3  0.15 ± 0.02e  0.07 ± 0.01e  12.78 ± 0.77d  0.34 ± 0.05e  0.08 ± 0.01c  0.86 ± 0.11c  12.36 ± 0.44c  L4  0.13 ± 0.02e  0.05 ± 0.01e  10.36 ± 0.71d  0.27 ± 0.03e  0.07 ± 0.01c  1.02 ± 0.13c  14.06 ± 0.62c  NtCYP71D16-Ri  L1  8.27 ± 0.51a  2.04 ± 0.21a  8.36 ± 0.52d  1.35 ± 0.15d  0.09 ± 0.01c  0.81 ± 0.12c  11.74 ± 0.40c  L3  11.09 ± 0.78a  3.17 ± 0.25a  5.97 ± 0.46d  1.17 ± 0.11d  0.08 ± 0.01c  0.74 ± 0.11c  12.14 ± 0.44c  L4  8.51 ± 0.52a  2.15 ± 0.21a  9.85 ± 0.61d  1.64 ± 0.16d  0.07 ± 0.01c  0.71 ± 0.11c  13.72 ± 0.61c    α-CBT-ol (μg cm−2)  β-CBT-ol (μg cm−2)  α-CBT-diol (μg cm−2)  β-CBT-diol (μg cm−2)  CBT-diol isomer (μg cm−2)  Sugar ester (μg cm−2)  Gibberellin (ng g−1)  CK  0.31 ± 0.05d  0.12 ± 0.02d  20.84 ± 1.01c  10.61 ± 0.32c  1.24 ± 0.14b  1.47 ± 0.16b  19.23 ± 0.94b  35S:NtCBTS2b  L1  0.74 ± 0.07c  0.24 ± 0.02c  31.57 ± 1.47b  17.56 ± 0.61b  1.85 ± 0.17a  2.08 ± 0.21a  25.67 ± 1.21a  L3  0.84 ± 0.08c  0.20 ± 0.02c  29.24 ± 1.35b  16.94 ± 0.57b  1.77 ± 0.16a  2.06 ± 0.20a  24.35 ± 1.16a  L4  0.67 ± 0.07c  0.21 ± 0.03c  32.94 ± 1.56b  18.74 ± 0.67b  1.92 ± 0.18a  2.15 ± 0.22a  26.34 ± 1.37a  CYP:NtCBTS2b  L1  2.34 ± 0.22b  0.64 ± 0.07b  44.28 ± 1.87a  27.67 ± 1.12a  1.95 ± 0.18a  2.16 ± 0.21a  24.39 ± 1.18a  L3  2.61 ± 0.27b  0.77 ± 0.08b  42.37 ± 1.71a  27.19 ± 1.10a  1.89 ± 0.18a  2.20 ± 0.24a  26.15 ± 1.34a  L4  2.48 ± 0.23b  0.67 ± 0.07b  46.7 1± 1.92a  28.63 ± 1.18a  2.07 ± 0.19a  2.24 ± 0.25a  26.75 ± 1.38a  NtCBTS2-Ri  L1  0.10 ± 0.02e  0.04 ± 0.01e  9.44 ± 0.60d  0.28 ± 0.03e  0.07 ± 0.01c  0.77 ± 0.10c  13.24 ± 0.53c  L3  0.15 ± 0.02e  0.07 ± 0.01e  12.78 ± 0.77d  0.34 ± 0.05e  0.08 ± 0.01c  0.86 ± 0.11c  12.36 ± 0.44c  L4  0.13 ± 0.02e  0.05 ± 0.01e  10.36 ± 0.71d  0.27 ± 0.03e  0.07 ± 0.01c  1.02 ± 0.13c  14.06 ± 0.62c  NtCYP71D16-Ri  L1  8.27 ± 0.51a  2.04 ± 0.21a  8.36 ± 0.52d  1.35 ± 0.15d  0.09 ± 0.01c  0.81 ± 0.12c  11.74 ± 0.40c  L3  11.09 ± 0.78a  3.17 ± 0.25a  5.97 ± 0.46d  1.17 ± 0.11d  0.08 ± 0.01c  0.74 ± 0.11c  12.14 ± 0.44c  L4  8.51 ± 0.52a  2.15 ± 0.21a  9.85 ± 0.61d  1.64 ± 0.16d  0.07 ± 0.01c  0.71 ± 0.11c  13.72 ± 0.61c  CK, non-transformed tobacco plants; 35S:NtCBTS2b. constitutive overexpression of NtCBTS2b; CYP:NtCBTS2b, overexpressing NtCBTS2b specifically in trichomes; NtCBTS2-Ri, suppression of the NtCBTS2 gene by RNAi; NtCYP71D16-Ri, suppression of the NtCYP71D16 gene by RNAi. Values are means ± SE (n = 50). a–eValues followed by different letters were significantly different at P <0.05. Fig. 6 View largeDownload slide Gas chromatographic analyses of the cembranoid contents in transgenic tobacco plants. Fifty leaf discs were punched from leaves, and analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. Fig. 6 View largeDownload slide Gas chromatographic analyses of the cembranoid contents in transgenic tobacco plants. Fifty leaf discs were punched from leaves, and analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. Constitutive expression of NtCBTS2b led to cembratrien-ol production in leaf mesophylls The precursor of cembranoid biosynthesis (GGPP) can be synthesized in all plant tissues, while cembranoid secretions are only produced in gland cells, and not detected in leaf mesophylls. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls using bioreactor engineering, the mesophylls of 35S:NtCBTS2b transgenic tobacco were used to analyze cembranoid contents. As predicted, CBT-ols and CBT-diols did not exist in the mesophylls of non-transformed tobacco plants (controls), while lots of CBT-ols and no CBT-diols were observed in the mesophylls of 35S:NtCBTS2b tobacco (Fig. 7). Moreover, there was no significant difference between 35S:NtCBTS2b tobacco and controls in terms of Chls, carotenoids, ABA and gibberellins (data not shown). Therefore, constitutive expression of NtCBTS2b may be a practical method to harvest large amounts of cembratrien-ols using genetic engineering. Fig. 7 View largeDownload slide Production of CBT-ols in mesophylls. (a) Gas chromatographic analyses of the cembranoid contents in the mesophylls of 35S:NtCBTS2b tobacco plants. Fifty leaf discs were punched from leaves and dipped in methylene chloride eight times to remove leaf exudates. The residual leaves without leaf exudates were analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. (b) Chart of the CBT-ols in the mesophylls of 35S:NtCBTS2b tobacco plants quantified by GC-MS. Fig. 7 View largeDownload slide Production of CBT-ols in mesophylls. (a) Gas chromatographic analyses of the cembranoid contents in the mesophylls of 35S:NtCBTS2b tobacco plants. Fifty leaf discs were punched from leaves and dipped in methylene chloride eight times to remove leaf exudates. The residual leaves without leaf exudates were analyzed by GC-MS for the cembranoid contents. Only the period from 35 to 45 min of the chromatogram is shown. (b) Chart of the CBT-ols in the mesophylls of 35S:NtCBTS2b tobacco plants quantified by GC-MS. Aphid infestation In previous studies, a CYP450-encoding gene (NtCYP71D16) specific to the trichome gland was identified, and the suppression of NtCYP71D16 led to a raised CBT-ol:CBT-diol ratio, which in turn promoted aphidicidal activity (Wang et al. 2001, Wang et al. 2004). To determine whether the promoted aphidicidal activity was due to an increase in CBT-ol production or the reduced attraction of CBT-diols, transgenic N. tabacum lines with different levels of CBT-ols or CBT-diols, labeled 35S:NtCBTS2b, NtCBTS2-Ri, CYP:NtCBTS2b and NtCYP71D16-Ri, were created using methods described previously (Wang et al. 2001) (Supplementary Fig. S2). In the bioactivity assay, the trichome exudates of wild-type and transgenic lines were individually applied on the leaf discs of wild-type tobacco (Fig. 8). Compared with controls, the number of aphids was more on the leaf discs of NtCBTS-Ri lines, fewer on 35S:NtCBTS2b lines and marginal on CYP:NtCBTS2b and NtCYP71D16-Ri lines. It was suggested that accumulation of CBT-ols may help to reduce aphid attraction. In accordance with the bioactivity assay, these transgenic lines had different levels of aphid resistance. As shown in Fig. 9, compared with controls, CYP:NtCBTS2b lines had enhanced CBT-ol and CBT-diol levels, while NtCYP71D16-Ri lines possessed increased CBT-ols and decreased CBT-diol levels. Notably, CYP:NtCBTS2b and NtCYP71D16-Ri lines were equally resistant to aphid colonization, and were more resistant than control lines. Resistance in the 35S:NtCBTS2b lines was also higher than in control lines, whereas it was lower than in CYP:NtCBTS2b and NtCYP71D16-Ri lines. It was noteworthy that the heaviest infestation was observed in NtCBTS-Ri lines, which had the lowest CBT-ol levels. These results indicated that accumulation of CBT-ols helped to elevate the aphid resistance of plants, and the level of CBT-diol was not related to the aphidicidal activity. Fig. 8 View largeDownload slide Bioactivity assays of the trichome exudates of wild-type and transgenic lines. (a) Colonization responses of aphids on the trichome exudates of wild-type and transgenic lines. (b) Aphid numbers on the wild-type leaf discs with different trichome exudates. Values are means ± SE (n = 10). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 8 View largeDownload slide Bioactivity assays of the trichome exudates of wild-type and transgenic lines. (a) Colonization responses of aphids on the trichome exudates of wild-type and transgenic lines. (b) Aphid numbers on the wild-type leaf discs with different trichome exudates. Values are means ± SE (n = 10). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 9 View largeDownload slide Aphid infestation response on different levels of CBT-ols. (a) Colonization responses of aphids on NtCBTS2b transgenic lines, NtCYP71D16-Ri lines and controls. (b) Survival of aphids on transgenic lines and controls. Values are means ± SE (n = 20). a–dValues followed by different letters are significantly different at P < 0.05. Fig. 9 View largeDownload slide Aphid infestation response on different levels of CBT-ols. (a) Colonization responses of aphids on NtCBTS2b transgenic lines, NtCYP71D16-Ri lines and controls. (b) Survival of aphids on transgenic lines and controls. Values are means ± SE (n = 20). a–dValues followed by different letters are significantly different at P < 0.05. Discussion The results of our study clarify our objectives, indicating that (i) NtCBTS2b plays a pivotal role in CBT-ol biosynthesis; NtCBTS2b overexpression promotes sugar ester and cembranoid production in glandular trichome cells; and accumulating CBT-ols promote aphid resistance; (ii) overexpression of NtCBTS2b may substantially promote the growth and development of glandular cells; and (iii) constitutive expression of NtCBTS2b led to cembratrien-ol production also in mesophylls, not only in glandular trichomes. Our data also indicate that NtCBTS is not transcribed and translated in N. tomentosiformis. Finally, our results confirm that NtCBTS2b is distributed in chloroplasts, confirming a notion long believed that, to date, had not yet been verified. Together, these data provide crucial insights into the biological function of NtCBTS2b in N. tabacum. As an important economic crop with large yields, tobacco leaves are harvested for a wide range of applications in the medical, food and biofuel industries. Tobacco-derived cembranoids are known to show antifungal, antibacterial, anti-human immunodeficiency virus, antitumor and neuroprotective properties (Zubair et al. 2014, Duan et al. 2015, Martins et al. 2015, Ning et al. 2017), while cembranoid secretions are only produced in glandular trichomes, and thereby the quantity produced is limited. Considering that the precursor of cembranoid biosynthesis (GGPP) can be synthesized in all plant tissues, it was possible to engineer a platform for cembratrien-ol production in leaf mesophylls through expressing NtCBTS2 in leaf mesophylls. Our results suggested that it was feasible to produce natural product cembratrien-ols in tobacco mesophylls using constitutive expression of NtCBTS2b. In future cembratrien-diol synthetic biology studies, the focus could be on the simultaneously constitutive expression of NtCBTS2b and NtCYP71D16 using genetic engineering. Our results demonstrate that overexpression or knockdown of NtCBTS2b did not suppress growth and development in N. tabacum. Indeed, the overexpression of NtCBTS2b substantially promotes the growth and development of glandular cells. One explanation for this increase in growth and development may be that the gibberellin concentration increased in NtCBTS2b-overexpressing lines. This result suggests that NtCBTS2b may be overexpressed in commercial plants with few or no adverse effects on plant growth, and that such overexpression promotes the production of such economically important compounds as cembranoids. Additionally, accumulation of CBT-ols promotes aphid resistance. Overexpressing NtCBTS2b specifically in glandular trichomes had similar aphidicidal activity to suppressing NtCYP71D16, even though the level of CBT-ols in NtCYP71D16-Ri lines was higher than that in CYP:NtCBTS2b lines. Noticeably, NtCYP71D16-suppressed transgenic lines produced less of the primary trichome exudations (CBT-diol), and produced more of its precursor (CBT-ol) instead. Previous studies have reported that the CBT-diols produced by tobacco plants inhibit growth in Valsa mali (Duan et al. 2015, Yan et al. 2017). Thus, overexpressing NtCBTS2b specifically in glandular trichomes may be an ideal method for promoting aphid resistance in tobacco taxa. Our study primarily focused on morphological traits, physiological parameters and aphid resistance in NtCBTS2b transgenic N. tabacum lines. We conclude that NtCBTS2b plays a key role in CBT-ol biosynthesis; CBT-ols plays important roles in aphid resistance; constitutive expression of NtCBTS2b leads to cembratrien-ol production in mesophylls rather than only in glandular trichomes; and overexpressing NtCBTS2b specifically in glandular trichomes is greatly important for promoting the aphid resistance of plants. Materials and Methods Plant materials Nicotiana tabacum ‘Kentucky 326’ plants were cultured in a greenhouse with a light/dark cycle of 14/10 h at 30/24°C. At the flowering stage, glandular trichomes, leaves with trichomes, leaves lacking trichomes, flowers, stems and roots were excised. Leaf trichomes were obtained by freezing leaves (length: 15 cm) on an aluminum sheet over a liquid nitrogen bath. Once frozen, a paintbrush was used to collect trichomes by brushing, and remaining leaf tissues were sampled as leaves lacking trichomes. Total RNA was isolated using TRIzol reagent (TAKARA), and then treated with RNase-free DNase I (TAKARA). Sequence analysis of full-length NtCBTS cDNA To obtain the coding region sequence of NtCBTS2, two primers were designed based on the lateral flanking sequences of the coding region. NCBI (https://www.ncbi.nlm.nih.gov/) and SGN (http://solgenomics.net/) databases were searched using BLAST to identify matches between deduced nucleotide and amino acid sequences and registered sequences. Sequence alignment was carried out using megAlign in DNAStar. The PROSITE database (http://expasy.hcuge.ch/sprot/prosite.html) was used to identify possible activity sites and functional regions. A total of 18 primers were used throughout all genetic analyses (Supplementary Table S1). NtCBTS2 expression patterns Semi-quantitative reverse transcription–PCR (RT–PCR) analysis and quantitative real-time PCR (qRT-PCR) were performed to detect the mRNA transcription levels of NtCBTS2 in different tobacco tissues. Expression of the ribosomal protein gene L25 (Volkov et al. 2003) was used as internal standard. Specific primers were designed based on sequence variations in NtCBTS2a and NtCBTS2b (Supplementary Table S1). qRT-PCR was performed to detect NtCBTS2 transcription levels in transgenic tobacco tissues. qRT-PCR was carried out with an ABI PRISM 7000 system (Applied Biosystems). Transcription levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001). Subcellular localization The coding sequence of NtCBTS2b was inserted upstream of GFP and downstream of 35S in the pJIT163-GFP vector for constructing an NtCBTS2b–GFP fusion protein. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Positive clones were infiltrated into tobacco leaves with a 1 ml syringe. Epidermal cells of infiltrated leaves were observed for green fluorescence with a laser-scanning confocal microscope (Leika TCS-NT; Leika) following 48 h incubation. Generation of transgenic plants Reverse and forward genetic approaches are commonly used to discern pivotal molecular factors. To determine the precise role of CBT-ols in N. tabacum, transgenic lines were generated in which NtCBTS2b was either overexpressed or silenced by RNA interference. Phenotypes of these different transgenic lines were characterized at different developmental stages. To test the effects of NtCBTS2b overexpression, the coding region of NtCBTS2b was separately inserted downstream of the constitutive CaMV 35S promoter in a pCAMBIA 1301 plasmid (Cambia), and downstream of a NtCYP450 trichome-specific promoter (Wang et al. 2002) in a modified pCAMBIA 1391 plasmid, yielding the p35S:NtCBTS2b and pCYP:NtCBT2Sb constructs, respectively. To test the effects of NtCBTS2 suppression, a 287 bp fragment of the NtCBTS2 coding sequence was ligated into the pK7GWIWG2(II) expression vector (Karimi et al. 2002). The recombinant vectors were separately transformed into tobacco using A. tumefaciens strain EHA105. Gene expression in NtCBTS2b transgenic lines For each construct, 15 independent transgenic lines were generated. T3 homozygous lines were selected based on hygromycin segregation in T1 and T2 seeds tested using χ2 tests for further analysis. The successful integration of recombinant vector into the genomes of transgenic lines was determined using DNA-PCR (Supplementary Fig. S3). To detect the effects of NtCBTS2b overexpression and knockdown in transgenic plants, four transgenic lines were used for the measurement of gene expression and protein abundance levels. Three control lines, i.e. N. tabacum ‘Kentucky 326’ and its diploid genome donors (N. tomentosiformis and N. sylvestris), were also used in qPCR and Western blotting analyses. NtCBTS protein level assays Proteins were isolated and quantified from approximately 0.1 g of four-leaf-old tobacco seedlings as described previously (Bradford 1976). A 20 μg aliquot of protein from each sample was electrophoretically fractioned in a 12.5% polyacrylamide gel and transferred onto a nitrocellulose membrane. Membranes were blotted using an NtCBTS2 polyclonal antibody (1 : 5,000; Abmart) and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1,000). The membrane was exposed to X-ray film (Fujifilm) to detect signals. Trichome morphology observations Seedlings were stained using 2% rhodamine at 37°C for 2 h, and then rinsed with ddH2O. Thirty seedling leaves of identical age and position were detached from transgenic plants (3 weeks old). Trichomes were photographed with an Axioplan 2 microscope (Carl Zeiss AG). Leaf trichom density, the area of glandular cells and the length and width of each trichome were recorded. Analysis of leaf surface exudates and isoprenoid contents Isoprenoid contents in transgenic lines were determined using methods described previously (Zhang et al. 2015). Gibberellin, ABA, IAA and zeatin riboside contents were measured using enzyme-linked immunosorbent assays (ELISAs). To isolate diterpenoid compounds produced in leaves, 50 leaf discs (diameter: 2 cm) were excised from leaves (leaf width: 10 cm) and immersed in 600 ml of methylene chloride for 2 s eight times. Leaf surface exudates were analyzed using gas chromatography–mass spectrometry (GC-MS). To detect whether diterpenoids are produced in the mesophylls of 35S:NtCBTS2b transgenic tobacco plants, these residual leaves after removing leaf exudates were pre-frozen in liquid nitrogen, and ground to a powder to analyze CBT-ols and CBT-diols. To measure carotenoid and Chl contents, 0.1 g of trichomes or 0.2 g of leaves were powdered and extracted with 25 ml of 90% acetone using ultrasonic extraction under shading conditions. Supernatants were collected and filtered through a 0.45 μm PTFE membrane filter. Pigment extracts were determined using reverse-phase HPLC. Bioactivity assays Fifty leaf discs (diameter: 2 cm) of wild-type or transgenic lines (35S:NtCBTS2b, CYP:NtCBTS2b, NtCBTS2-Ri and NtCYP71D16-Ri) were excised from leaves (leaf width: 10 cm) to collect trichome exudates as described previously (Zhang et al. 2015). Wild-type tobacco plants were cultured for 4 weeks, and leaf discs (diameter: 5 cm) were excised from leaves (leaf width: 10 cm). Ten leaf discs were immersed in the trichome exudate solution extracted from each transgenic line for 5 s, and placed in a closed container at room temperature. Leaf discs were arranged evenly in a circle. Two hundred aphids were released in the center of the circle, and infested these leaf discs naturally. The number of aphids on each leaf disc was counted 2 d later. Bioactivity assays were carried out in triplicate. Aphid infestation The tobacco aphid (Myzus nicotianae), a major tobacco pest with a cosmopolitan distribution, was used in the assay. Transgenic seeds were germinated and cultured in a greenhouse with a light/dark cycle of 14/10 h at 30/24°C. Three weeks later, 1,000 aphids were released and were allowed to infest the plants naturally. The number of aphids infesting each transgenic line was scored after 2 weeks. Aphid infestation experiments were carried out in triplicate. Data analysis All statistical procedures were conducted using the Student’s t-test in SAS v9.2 to detect significant differences between means. The significance threshold was set at 0.05 or 0.01. Supplementary Data Supplementary data are available at PCP online. Funding The work was supported by the State Tobacco Monopoly Administration of China [grant No. 110201401003 (JY-03)]. Disclosures The authors have no conflicts of interest to declare. References Bradford M.M. ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem.  72: 248– 254. Google Scholar CrossRef Search ADS PubMed  Bruckner K., Tissier A. ( 2013) High-level diterpene production by transient expression in Nicotiana benthamiana. Plant Methods  9: 46. Google Scholar CrossRef Search ADS PubMed  Cheng A.X., Lou Y.G., Mao Y.B., Lu S., Wang L.J., Chen X.Y. 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Abbreviations Abbreviations CBT-diol cembratrienediol CBT-ol cembratrieneol CBTS cembratrien-ol synthase CYP promoter CYP71D16 trichome-specific promoter CYP450 Cyt P450 oxygenase CYP:NtCBTS2b transgenic plants expressing NtCBTS2b under the control of the CYP promoter GC-MS gas chromatography–mass spectrometry GFP green fluorescent protein GGPP geranylgeranyl diphosphate qRT-PCR quantitative real-time PCR RNAi RNA interference RT–PCR reverse transcription–PCR 35S Cauliflower mosaic virus (CaMV) 35S promoter 35S:NtCBTS2b transgenic plants expressing NtCBTS2b under the control of the 35S promoter NtCBTS2-Ri transgenic plants with NtCBTS2 suppressed by RNA interference © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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