Abstract

   Nicotiana tabacum solanesyl diphosphate synthase 1 (NtSPS1) is the key
   enzyme in solanesol biosynthesis. However, changes in the solanesol
   content, plant growth, photosynthesis, and metabolome of tobacco plants
   after NtSPS1 overexpression (OE) have not been previously reported. In
   the present study, these parameters, as well as photosynthetic gas
   exchange, chlorophyll content, and chlorophyll fluorescence parameters,
   were compared between NtSPS1 OE and wild type (WT) lines of tobacco. As
   expected, NtSPS1 OE significantly increased solanesol content in
   tobacco leaves. Although NtSPS1 OE significantly increased leaf growth,
   photosynthesis, and chlorophyll content, the chlorophyll fluorescence
   parameters in the leaves of the NtSPS1 OE lines were only slightly
   higher than those in the WT leaves. Furthermore, NtSPS1 OE resulted in
   64 differential metabolites, including 30 up-regulated and 34
   down-regulated metabolites, between the OE and WT leaves. Pathway
   enrichment analysis of these differential metabolites identified
   differentially enriched pathways between the OE and WT leaves, e.g.,
   carbon fixation in photosynthetic organisms. The maximum carboxylation
   rate of RuBisCO and the maximum rate of RuBP regeneration were also
   elevated in the NtSPS1 OE line. To our knowledge, this is the first
   study to confirm the role of NtSPS1 in solanesol biosynthesis and its
   possible functional mechanisms in tobacco.

   Keywords: Nicotiana tabacum, solanesol, solanesyl diphosphate synthase,
   plant growth, photosynthesis, metabolome

1. Introduction

   Solanesol, a non-cyclic polyisoprenoid alcohol with nine isoprene
   units, has antioxidant, anti-inflammatory, antimicrobial, and
   neuroprotective activities [[34]1,[35]2,[36]3,[37]4]. Recently, the
   neuroprotective effects of solanesol from behavioural and biochemical
   perspectives in the intracerebroventricular propionic acid induced
   experimental model of autism have been studied [[38]4]. Solanesol also
   serves as an important pharmaceutical intermediate in the chemical
   synthesis of ubiquinone drugs, including the anticancer agent
   synergiser N-solanesyl-N,N′-bis(3,4-dimethoxybenzyl) ethylenediamine
   (SDB), vitamin K2, and coenzyme Q10 [[39]1,[40]5,[41]6]. Among them,
   SDB can inhibit P-glycoprotein-mediated multidrug resistance and
   thereby be used to reverse drug resistance of the paclitaxel-resistant
   cell line (KK47/TX30) [[42]7,[43]8]. The lipid-soluble vitamin K2 has
   positive effects on the treatment of osteoporosis, promotion of blood
   clotting, and inhibition of vascular calcification [[44]9]. The
   lipid-soluble coenzyme Q10 participates in ATP synthesis and oxidative
   phosphorylation, and it acts as an activator of cellular respiration
   and cellular metabolism [[45]10]. Moreover, coenzyme Q10 can be used
   for the treatment of neurodegenerative diseases, renal failure, and
   cardiovascular diseases [[46]11]. Coenzyme Q10 and solanesyl
   poly(ethylene glycol) succinate were formulated as micelles to improve
   the bioavailability of the former in rats [[47]12]. Moreover, solanesyl
   poly(ethylene glycol) dithiodipropionate and solanesyl thiosalicylic
   acid micelles were found to serve as efficient drug carriers with
   synergistic anticancer effects [[48]13,[49]14]. Therefore, the
   pharmaceutical value of solanesol and its derivatives has been widely
   confirmed.

   Solanesol mainly exists in solanaceous crops, such as tomato (Solanum
   lycopersicum), potato (Solanum tuberosum), and tobacco (Nicotiana
   tabacum) [[50]1,[51]5,[52]6,[53]15,[54]16], with N. tabacum possessing
   the highest content [[55]1,[56]5,[57]6]. As solanesol is synthesized in
   the chloroplast, it mainly accumulates in the leaves and other green
   tissues [[58]15]. However, its accumulation is also affected by
   environmental and genetic factors [[59]1,[60]17,[61]18]. Moderately
   high temperatures [[62]16], pathogen infection [[63]19],
   long-wavelength/extended irradiation, rare-earth elements, and shade
   [[64]5] are some of the environmental factors that can lead to
   increases in the solanesol content of tobacco leaves. In terms of
   genetic factors, the solanesol content in potato leaves was found to be
   controlled by quantitative trait genes [[65]18], and that in tobacco
   leaves was found to be determined by both polygenes and major genes,
   but the major genes were dominant [[66]20].

   Solanesol is synthesized via the methylerythritol 4-phosphate (MEP)
   pathway in plastids [[67]5,[68]6,[69]15,[70]16]. The synthesis of
   solanesyl diphosphate from C5 isopentenyl diphosphate and dimethylallyl
   diphosphate, and the direct precursors (C10 geranyl diphosphate, C15
   farnesyl diphosphate, and C20 geranylgeranyl diphosphate) is catalyzed
   by solanesyl diphosphate synthase (SPS) and represents a key step in
   solanesol synthesis ([71]Figure S1) [[72]6]. To date, SPS genes have
   been identified in Arabidopsis thaliana [[73]21], Oryza sativa
   [[74]22], S. lycopersicum [[75]23], and N. tabacum [[76]24]. Silencing
   of AtSPS1 and AtSPS2 in A. thaliana lowered leaf plastoquinone content,
   thus reducing photosynthesis and inducing photoinhibition [[77]25]. In
   O. sativa, OsSPS1 and OsSPS2 preferentially catalyze the synthesis of
   ubiquinone-9 and plastoquinone-9 in mitochondria and plastids,
   respectively [[78]22]. Overexpression (OE) of tomato SPS in tobacco
   significantly increased the plastoquinone content of immature leaves
   [[79]23]. The expression levels of NtSPS1 in different organs of
   tobacco plants decreased according to this order: leaf > stem > root,
   which was consistent with the distribution of solanesol in tobacco
   plants [[80]24]. Studies on SPS function have mainly been conducted on
   A. thaliana, rice, tomato, and other model crops, while the function of
   NtSPS genes in tobacco, the plant with the highest solanesol content,
   has not been reported.

   Leaves are the main organs of solanesol accumulation
   [[81]5,[82]15,[83]17,[84]18] and are also the most active plant tissues
   for photosynthesis. Chloroplasts are abundant in mesophyll cells, and
   chloroplasts contain chlorophyll, a green pigment that can absorb light
   energy [[85]26]. During photosynthesis, plants use light to oxidize
   water and release oxygen and to reduce carbon dioxide (CO[2]) to
   synthesize a large number of carbohydrates [[86]27,[87]28].
   Photosynthetic gas exchange and chlorophyll fluorescence parameters are
   the most commonly used phenotypic indicators of plant growth and
   photosynthesis [[88]29,[89]30]. Moreover, photosynthesis provides the
   initial substrate and energy for the synthesis of solanesol and other
   secondary metabolites [[90]16]. Metabolites are the basis of biological
   phenotypes, which can help us understand biological processes and
   mechanisms more intuitively and effectively [[91]31]. To date, the
   changes in solanesol content, plant growth, photosynthesis, and the
   metabolome of tobacco leaves after NtSPS1 OE have not been reported.
   Thus, we compared the differences in solanesol content, plant growth,
   photosynthetic gas exchange, chlorophyll content, chlorophyll
   fluorescence parameters, and the metabolome between NtSPS1 OE and wild
   type (WT) lines of tobacco plants. The purpose of this study was to
   analyze the role of NtSPS1 in solanesol biosynthesis and its possible
   functional mechanisms to provide a theoretical basis for the regulation
   of solanesol content in tobacco leaves by means of genetic engineering.

2. Results

2.1. Total Solanesol Content and NtSPS1 Expression Levels in the Leaves of
NtSPS1 OE and WT Tobacco Plants

   Eighteen NtSPS1 transgenic plants were obtained, 16 of which were
   finally confirmed to be positive ([92]Figure S2). Of these 16
   independently derived transgenic lines, five showed NtSPS1 transcript
   levels that were >600% WT levels, and the three lines (OE-#1, OE-#2,
   and OE-#5) with the greatest OE of NtSPS1 transcripts were investigated
   further in this study ([93]Figure 1). The total solanesol content in
   the leaves of OE-#1, OE-#2, and OE-#5, was 89.62%, 77.05%, and 64.48%
   higher than that in the WT, respectively ([94]Figure 1A), and the
   relative NtSPS1 transcript levels in the leaves were 8.80, 7.97, and
   7.68 times those of the WT, respectively ([95]Figure 1B). Moreover, the
   relative NtSPS1 transcript levels in the leaves of OE-#1 were
   significantly higher than those in the leaves of OE-#2 and OE-#5 (p <
   0.05). Thus, OE-#1 was selected as the representative NtSPS1 OE line
   for further examination.

Figure 1.

   [96]Figure 1
   [97]Open in a new tab

   Identification of NtSPS1 overexpression (OE) tobacco lines. Total
   solanesol content (A) and relative NtSPS1 transcript levels (B) in the
   leaves of three NtSPS1 OE and wild type (WT) tobacco plants. Different
   letters on the columns indicate p < 0.05. DW, dry weight.

   We measured the total solanesol content and relative NtSPS1 transcript
   levels of the OE and WT tobacco leaves 0–12 days after sampling (DAS).
   As the leaves grew, their total solanesol content in the NtSPS1 OE and
   WT tobacco leaves increased from 0 to 12 DAS ([98]Figure 2A). At 0, 3,
   6, 9, and 12 DAS, the total solanesol content in the leaves of the
   NtSPS1 OE tobacco plants increased by 93.75%, 109.26%, 112.50%,
   138.30%, and 132.92%, respectively, and these contents were
   significantly higher than those in the WT tobacco leaves (p < 0.05)
   ([99]Figure 2A). At 0, 3, 6, 9, and 12 DAS, the relative NtSPS1
   transcript levels in the leaves of the NtSPS1 OE tobacco leaves
   increased 8.21, 8.06, 7.90, 8.02, and 8.52 times, respectively, and
   were significantly higher than the corresponding levels in the WT
   tobacco leaves (p < 0.05) ([100]Figure 2B). Thus, NtSPS1 OE
   significantly increased total solanesol content in the tobacco leaves.

Figure 2.

   [101]Figure 2
   [102]Open in a new tab

   Total solanesol content (A) and relative NtSPS1 transcript levels (B)
   in the leaves of NtSPS1 overexpression (OE) and wild type (WT) tobacco
   plants. An asterisk (*) indicates p < 0.05 versus the corresponding WT
   value. DW, dry weight.

2.2. Growth of the NtSPS1 OE and WT Tobacco Plants

   To determine the effects of NtSPS1 OE on growth of the tobacco plants,
   we measured the plant height, leaf length, leaf width, and leaf dry
   weight of the NtSPS1 OE and WT tobacco plants 0–12 DAS. At 0, 3, 6, 9,
   and 12 DAS, the plant heights of the NtSPS1 OE tobacco plants increased
   by 20.20%, 18.61%, 16.22%, 17.86%, and 16.81%, respectively, and these
   heights were significantly higher than those of the WT plants (p <
   0.05) ([103]Figure 3A). At 0, 3, 6, 9, and 12 DAS, the leaf lengths of
   the NtSPS1 OE tobacco plants increased by 33.47%, 40.83%, 34.69%,
   31.26%, and 32.21%, respectively, values that were significantly higher
   than those of their WT counterparts (p < 0.05) ([104]Figure 3B).
   Similarly, at 0, 3, 6, 9, and 12 DAS, the leaf widths of the NtSPS1 OE
   tobacco plants increased significantly (by 26.13%, 32.71%, 30.52%,
   21.18%, and 20.44%, respectively) compared with the corresponding
   values of the WT plants (p < 0.05) ([105]Figure 3C). As expected, the
   leaf dry weight of the NtSPS1 OE and WT tobacco plants increased 0–12
   DAS ([106]Figure 3D). At 0, 3, 6, 9, and 12 DAS, the leaf dry weights
   of the NtSPS1 OE tobacco plants increased by 32.79%, 31.43%, 35.37%,
   37.89%, and 36.36%, respectively, and these weights were significantly
   higher than those of the WT plants (p < 0.05) ([107]Figure 3D). Thus,
   NtSPS1 OE significantly promoted the growth of tobacco plants and
   leaves, as reflected by the increased plant height, leaf length, leaf
   width, and leaf dry weight of the NtSPS1 OE tobacco plants.

Figure 3.

   [108]Figure 3
   [109]Open in a new tab

   Plant height (A), leaf length (B), leaf width (C), and leaf dry weight
   (D) in the NtSPS1 overexpression (OE) and wild type (WT) tobacco
   plants. An asterisk (*) indicates p < 0.05 versus the corresponding WT
   value.

2.3. Photosynthetic Gas Exchange in the Leaves of NtSPS1 OE and WT Tobacco
Plants

   To determine the effects of NtSPS1 OE on the photosynthetic gas
   exchange of tobacco leaves, we measured the net photosynthetic rate
   (Pn), stomatal conductance (Gs), intercellular CO[2] concentration
   (Ci), and transpiration rate (Tr) in the leaves of the NtSPS1 OE and WT
   tobacco plants with a portable open gas exchange system. At 0, 3, 6, 9,
   and 12 DAS, the Pn of the NtSPS1 OE tobacco leaves increased by 29.14%,
   32.74%, 25.20%, 20.19%, and 17.54%, respectively, and these values were
   significantly higher than those of the WT tobacco leaves (p < 0.05)
   ([110]Figure 4A). At 0, 3, 6, 9, and 12 DAS, the Gs of the NtSPS1 OE
   tobacco leaves increased by 19.05%, 22.73%, 20.83%, 19.23%, and 17.86%,
   respectively, and these values were also significantly higher than
   those of the WT tobacco leaves (p < 0.05) ([111]Figure 4B). However, at
   0, 3, 6, 9, and 12 DAS, the Ci of the WT tobacco leaves increased by
   4.98%, 4.84%, 5.37%, 5.94%, and 8.06%, respectively, and these values
   were slightly, but not significantly, higher than those of the NtSPS1
   OE leaves (p > 0.05) ([112]Figure 4C). At 0, 3, 6, 9, and 12 DAS, the
   Tr of the NtSPS1 OE leaves increased by 14.68%, 12.95%, 13.58%, 12.92%,
   and 11.68%, respectively, and these values were significantly higher
   than those of the WT tobacco leaves (p < 0.05) ([113]Figure 4D). Thus,
   NtSPS1 OE significantly enhanced photosynthesis, as reflected by the
   increased Pn, Gs, and Tr in the NtSPS1 OE tobacco leaves.

Figure 4.

   [114]Figure 4
   [115]Open in a new tab

   Net photosynthetic rate (Pn, A), stomatal conductance (Gs, B),
   intercellular CO[2] concentration (Ci, C), and transpiration rate (Tr,
   D) in the leaves of NtSPS1 overexpression (OE) and wild type (WT)
   tobacco plants. An asterisk (*) indicates p < 0.05 versus the
   corresponding WT value.

2.4. Chlorophyll Content in the Leaves of NtSPS1 OE and WT Tobacco Plants

   To determine the effects of NtSPS1 OE on the chlorophyll content of
   tobacco leaves, we measured chlorophyll a and chlorophyll b content in
   the leaves of NtSPS1 OE and WT tobacco plants with a UV-2700 UV-VIS
   spectrophotometer. At 0, 3, 6, 9, and 12 DAS, the chlorophyll a content
   in the NtSPS1 OE tobacco leaves increased by 14.29%, 16.07%, 23.21%,
   16.95%, and 19.05%, respectively, and these values were significantly
   higher than those in the WT tobacco leaves (p < 0.05) ([116]Figure 5A).
   At 0, 3, 6, 9, and 12 DAS, the chlorophyll b content in the NtSPS1 OE
   tobacco leaves increased by 7.69%, 11.54%, 11.54%, 7.41%, and 10.71%,
   respectively, and these values were also significantly higher than
   those in the WT tobacco leaves (p < 0.05) ([117]Figure 5B). Thus,
   NtSPS1 OE significantly increased chlorophyll content, as reflected by
   the increased chlorophyll a and chlorophyll b levels in the NtSPS1 OE
   tobacco leaves.

Figure 5.

   [118]Figure 5
   [119]Open in a new tab

   Chlorophyll a (A) and chlorophyll b (B) content in the leaves of NtSPS1
   overexpression (OE) and wild type (WT) tobacco plants. An asterisk (*)
   indicates p < 0.05 versus the corresponding WT value.

2.5. Chlorophyll Fluorescence Parameters in the Leaves of NtSPS1 OE and WT
Tobacco Plants

   To determine the effects of NtSPS1 OE on the chlorophyll fluorescence
   parameters of tobacco leaves, we measured the maximum quantum
   efficiency of photosystem II (PSII) in dark-adapted leaves (F[v]/F[m]),
   quantum efficiency of PSII under light conditions (Φ[PSII]),
   photochemical quenching (qP), and electron transport rate (ETR) in the
   leaves of the NtSPS1 OE and WT tobacco plants with an Imaging-PAM-M
   series chlorophyll fluorometer. At 0, 3, 6, 9, and 12 DAS, the
   F[v]/F[m]of the NtSPS1 OE tobacco leaves increased by 2.67%, 2.67%,
   2.63%, 2.63%, and 2.63%, respectively ([120]Figure 6A), and the Φ[PSII]
   increased by 6.90%, 6.67%, 6.45%, 6.25%, and 6.25%, respectively
   ([121]Figure 6B), all slightly higher than the corresponding values in
   the WT tobacco leaves (p > 0.05). Furthermore, at 0, 3, 6, 9, and 12
   DAS, the qP of the NtSPS1 OE tobacco leaves increased by 5.88%, 5.77%,
   5.66%, 5.56%, and 5.56%, respectively, ([122]Figure 6C), and the ETR
   increased by 5.81%, 5.12%, 5.70%, 5.64%, and 5.54%, respectively
   ([123]Figure 6D), all slightly higher than the corresponding values in
   the WT tobacco leaves (p > 0.05). Thus, NtSPS1 OE slightly increased
   chlorophyll fluorescence parameters in tobacco leaves, as reflected by
   slightly increased F[v]/F[m], Φ[PSII], qP, and ETR in the NtSPS1 OE
   tobacco leaves, although these differences were not statistically
   significant.

Figure 6.

   [124]Figure 6
   [125]Open in a new tab

   Maximum quantum efficiency of photosystem II in the dark-adapted leaves
   (F[v]/F[m], (A)), quantum efficiency of photosystem II under light
   conditions (Φ[PSII], (B)), photochemical quenching (qP, (C)), and
   electron transport rate (ETR, (D)) in the leaves of the NtSPS1
   overexpression (OE) and wild type (WT) tobacco plants.

2.6. Leaf Metabolome of the NtSPS1 OE and WT Tobacco Plants

   In the present study, 477 metabolites were identified in the NtSPS1 OE
   and WT tobacco leaves with a QTRAP^® 6500+ mass spectrometer (AB SCIEX,
   Framingham, MA, USA), comprising two alcohols and polyols, 11 alkaloids
   and their derivatives, 74 amino acids and their derivatives, six
   benzene and substituted derivatives, six benzoic acid and substituted
   derivatives, 39 carbohydrates, one choline, 14 fatty acids, one
   flavanone, one flavone, one flavone C-glycoside, 15 flavonoids, two
   flavonols, one free fatty acid, four glycerophospholipids, two
   hydroxycinnamoyl derivatives, four indole and substituted derivatives,
   one iridoid glycoside, two ketones, one lactone, 13 lipids and
   lipid-like molecules, 13 nucleic acid derivatives, 36 nucleotides and
   their derivatives, 65 organic acids and their derivatives, seven
   organic oxygen compounds, three organoheterocyclic compounds, one
   phenolamide, eight phenolamines, one phenolic acid, seven phenol and
   substituted derivatives, 14 phenylpropanoids and polyketides, one
   phospholipid, seven phytohormones, 10 polyphenols, eight polyamines,
   four pyridine and substituted derivatives, two pyrimidines, two quinate
   and substituted derivatives, two sugar acids and their derivatives,
   four sugar alcohols, seven TCA cycle intermediates, nine traditional
   Chinese medicines, six terpenes, six terpenoids, two vitamin
   derivatives, 16 vitamins, and 35 other metabolites ([126]Table S1).
   Moreover, 64 metabolites were identified as differential metabolites
   between the NtSPS1 OE and WT leaves ([127]Table S2). Compared with the
   WT tobacco leaves, there were 30 up-regulated and 34 down-regulated
   metabolites in the NtSPS1 OE tobacco leaves ([128]Figure 7A; [129]Table
   S2).

Figure 7.

   [130]Figure 7
   [131]Open in a new tab

   Differential metabolite volcano plot (A) and Kyoto Encyclopedia of
   Genes and Genomes (KEGG) enrichment bubble chart (B) between the leaves
   of the NtSPS1 overexpression (OE) and wild type (WT) tobacco plants. In
   part A, the abscissa indicates the expression change (log2 fold change)
   in the metabolites in the different groups and the ordinate indicates
   differences in the significance levels (−log10 (p-value)). Each point
   in the volcano plot represents a metabolite and the size of the point
   indicates the variable importance in project (VIP) value. Significantly
   up-regulated metabolites are represented by red points, and
   significantly down-regulated metabolites are represented by green
   points. In part B, the abscissa indicates the number of differential
   metabolites in the corresponding metabolic pathway/the number of total
   metabolites identified in the pathway. The larger the value is, the
   higher the number of differential metabolites in the pathway. The color
   of the points represents the p-value of hypergeometric test. The
   smaller the value is, the more reliable and statistically significant
   the test is. The size of the point represents the number of
   differential metabolites in the corresponding pathway.

   To further clarify the metabolic pathways involved in the synthesis of
   these differential metabolites, we conducted Kyoto Encyclopedia of
   Genes and Genomes (KEGG) pathway enrichment analysis, which identified
   28 enriched metabolic pathways for the differential metabolites between
   NtSPS1 OE and WT tobacco leaves ([132]Table S3). Twenty of the most
   enriched pathways were selected for further study ([133]Figure 7B).
   Pathways that encompassed two differential metabolites included
   “phenylalanine metabolism,” “tyrosine metabolism,” “carbon fixation in
   photosynthetic organisms,” “tropane, piperidine, and pyridine alkaloid
   biosynthesis,” “tryptophan metabolism,” “amino sugar and nucleotide
   sugar metabolism,” “indole alkaloid biosynthesis,” “pantothenate and
   CoA biosynthesis,” and “pentose phosphate pathway”. The pathways that
   encompassed one differential metabolite included “starch and sucrose
   metabolism,” “beta-alanine metabolism,” “glycolysis/gluconeogenesis,”
   “sulfur relay system,” “ubiquinone and other terpenoid−quinone
   biosynthesis,” “phosphonate and phosphinate metabolism,” “taurine and
   hypotaurine metabolism,” “vancomycin resistance,” “vitamin B6
   metabolism,” “alpha−linolenic acid metabolism,” and “selenocompound
   metabolism”. Among these enriched metabolic pathways, the “carbon
   fixation in photosynthetic organisms” pathway is closely related to
   photosynthesis.

2.7. Maximum Carboxylation Rate of RuBisCO and Maximum Rates of RuBP
Regeneration in Leaves of the NtSPS1 OE and WT Tobacco Plants

   To determine the effects of NtSPS1 OE on carbon fixation in tobacco
   leaves, we used a mathematical model to calculate the maximum
   carboxylation rate of RuBisCO (Vc,max) and maximum rates of RuBP
   regeneration (Jmax) in the leaves of the NtSPS1 OE and WT tobacco
   plants. Both Vc,max and Jmax increased significantly in the NtSPS1 OE
   leaves (by 17.20% and 16.06%, respectively), compared with the
   corresponding value in the WT tobacco leaves (p < 0.05).

3. Discussion

3.1. NtSPS1 OE Enhances Solanesol Accumulation in Tobacco Leaves

   The solanesol content in tobacco leaves is influenced by both
   environmental and genetic factors [[134]17,[135]18], but genetic
   factors exert a considerable influence [[136]1]. To identify
   solanesol-rich tobacco varieties, Xiang et al. [[137]20] determined the
   total solanesol content in the leaves of 168 flue-cured tobacco sources
   from various years and regions in China and found that their solanesol
   content was between 0.70% and 4.13%. To analyze the influence of
   solanesol biosynthetic genes on solanesol accumulation in tobacco, Gai
   et al. [[138]32] measured solanesol content in the leaves, stems, and
   roots and quantified solanesol biosynthetic gene expression in the
   tobacco cultivars “Zhongyan90,” which has low solanesol content, and
   “Hongda,” which has high solanesol content. Their results indicated
   that the solanesol biosynthetic genes in both “Zhongyan90” and “Hongda”
   may regulate solanesol content through synergistic effects. In
   addition, transient OE of genes from the mevalonic acid and MEP
   pathways significantly increased solanesol content in the leaves of
   Nicotiana benthamiana [[139]18]. Similarly, overexpression of the
   1-deoxy-d-xylulose-5-phosphate reductoisomerase gene in chloroplasts
   increased the solanesol content in tobacco leaves [[140]33].
   Overexpressing the key enzyme gene of solanesol biosynthesis, NtSPS1,
   significantly increased the total solanesol content in tobacco leaves
   under day/night temperatures of 30/24 °C ([141]Figure 1 and [142]Figure
   2). In our previous study, moderately high temperatures (day/night
   temperatures of 30/24 °C) resulted in significantly higher solanesol
   content and NtSPS1 expression than normal temperatures (day/night
   temperatures of 22/16 °C), suggesting that the increased expression of
   NtSPS1 is related to the increase in solanesol content under moderately
   high temperatures [[143]16]. Solanesol is synthesized in chloroplasts
   [[144]5,[145]6,[146]15,[147]16]. We cannot exclude the possibility that
   the increase in total solanesol content in the NtSPS1 OE tobacco leaves
   was related to their increased chlorophyll content. Regardless, to the
   best of our knowledge, the present study is the first to confirm the
   role of NtSPS1 in solanesol biosynthesis in tobacco.

3.2. NtSPS1 OE Enhances Tobacco Plant Growth and Photosynthesis

   The leaf is the organ with the highest solanesol accumulation
   [[148]5,[149]15]. Thus, leaf biomass has a significant effect on the
   final yield of solanesol. The present study showed that NtSPS1 OE
   significantly increased the length, width, and dry weight of tobacco
   leaves ([150]Figure 3). Similarly, tobacco plants overexpressing
   H-protein showed higher leaf area and leaf dry weight than WT tobacco
   plants [[151]34]. In the present study, NtSPS1 OE increased the
   solanesol content in tobacco leaves ([152]Figure 1 and [153]Figure 2)
   and also increased the biomass of tobacco leaves, which thus greatly
   increased the final yield of solanesol from the entire tobacco plant.
   Similarly, OE of the small auxin-up RNA gene significantly increased
   the biomass and content of tropine alkaloids in Atropa belladonna
   [[154]35].

   The augmenting effect of NtSPS1 OE on tobacco leaf biomass is related
   to an increased Pn of the leaves ([155]Figure 4). Moreover, NtSPS1 OE
   may enhance Pn by increasing Gs or by affecting non-stomatal
   biochemical processes. The present study showed that the Gs of the
   NtSPS1 OE leaves was significantly higher than that of the WT tobacco
   leaves ([156]Figure 4). The increase in Tr in the NtSPS1 OE tobacco
   leaves was possibly related to the increase in Gs and corresponded with
   the stomatal aperture [[157]36]. In the present study, enhanced Gs in
   the NtSPS1 OE tobacco leaves was beneficial for CO[2] movement in the
   stomatal cavity. In addition, the increase in Pn caused by NtSPS1 OE
   was probably mediated through non-stomatal biochemical processes, such
   as elevations in chlorophyll content ([158]Figure 5) and ETR
   ([159]Figure 6). Similarly, OE of the cotton FLOWERING LOCUS T
   (FT)-like gene [[160]37] or Medicago sativa glutamate-semialdehyde
   aminotransferase gene [[161]38] led to increased chlorophyll content
   and photosynthesis efficiency in tobacco leaves. In rice, OE of the A.
   thaliana NAD kinase gene caused increased ETR and CO[2] assimilation
   rates [[162]39]. In A. thaliana, the OE of AtSPS1 can lead to the
   accumulation of plastoquinone-9 and its derivative plastochromanol-8 in
   the leaves and can reduce lipid peroxidation and PSII photoinhibition
   under excess light [[163]40]. In Salvia miltiorrhiza, the OE of
   polyprenyl diphosphate synthase 1 also significantly increased the
   content of plastoquinone-9, which is the main carrier of photosynthetic
   electrons [[164]41]. In our previous study, moderate/high temperatures
   resulted in significantly higher Pn and NtSPS1 expression in tobacco
   leaves than normal temperatures [[165]16]. The present study confirmed
   that NtSPS1 OE enhances the biomass and photosynthesis of tobacco
   leaves. Moreover, the increase in Pn in the NtSPS1 OE tobacco leaves
   augments initial substrate levels and energy for solanesol
   biosynthesis.

3.3. Effects of NtSPS1 OE on the Metabolome of Tobacco Leaves

   In the present study, NtSPS1 OE resulted in 64 differential metabolites
   between the NtSPS1 OE and WT leaves, including 30 up-regulated and 34
   down-regulated metabolites ([166]Figure 7A). In Brassica napus, OE of
   the lipid transfer protein BraLTP2 altered the accumulation of
   secondary metabolites in leaves, including 43 up-regulated and 30
   down-regulated secondary metabolites [[167]42]. The present study
   indicated that NtSPS1 OE not only enhanced solanesol accumulation in
   tobacco leaves but also altered many metabolic pathways in the leaves
   ([168]Figure 7B). Similarly, amorpha-4,11-diene synthase overexpression
   not only affects artemisinin biosynthesis but also affects the whole
   metabolic network of terpenoids in Artemisia annua [[169]43]. The KEGG
   pathway enrichment analysis indicated that 28 metabolic pathways were
   enriched to produce the differential metabolites between NtSPS1 OE and
   WT tobacco leaves. Among these metabolic pathways, carbon fixation in
   photosynthetic organisms [[170]44] is closely related to plant
   photosynthesis. Moreover, NtSPS1 OE may enhance carbon fixation by
   increasing ribose 5-phosphate content ([171]Tables S2 and S3), which
   affects CO[2] fixation in chloroplasts [[172]45]. The increased CO[2]
   fixation rate could result from alterations in only part of the carbon
   reduction cycle utilizing ATP from the photochemical reactions to
   convert ribose 5-phosphate to ribose 5-diphosphate (the carboxylation
   reaction substrate) [[173]46]. Therefore, the enhanced photosynthesis
   by NtSPS1 OE may be related to the ability of NtSPS1 OE to promote
   carbon fixation (Vc,max and Jmax) in tobacco leaves ([174]Table 1).
   Except for “carbon fixation in photosynthetic organisms,” the pathways
   that encompassed two differential metabolites between the NtSPS1 OE and
   WT tobacco leaves included “phenylalanine metabolism”; “tyrosine
   metabolism”; “tropane, piperidine, and pyridine alkaloid biosynthesis”;
   “tryptophan metabolism”; “amino sugar and nucleotide sugar metabolism”;
   “indole alkaloid biosynthesis”; “pantothenate and CoA biosynthesis”;
   and “pentose phosphate pathway” ([175]Figure 7B). Thus, KEGG pathway
   enrichment analysis of the differential metabolites identified enriched
   pathways between the NtSPS1 OE and WT tobacco leaves.

Table 1.

   Maximum carboxylation rate of RuBisCO (Vc,max) and maximum rates of
   RuBP regeneration (Jmax) in the leaves of NtSPS1 OE and WT tobacco
   plants 12 days after sampling. An asterisk (*) indicates p < 0.05
   versus the corresponding WT value.
      Vc,max (μmol·m^−2·s^−1) Jmax (μmol·m^−2·s^−1)
   WT       68.6 ± 3.9             153.8 ± 6.4
   OE      80.4 ± 4.1 *           178.5 ± 6.8 *
   [176]Open in a new tab

4. Materials and Methods

4.1. Plant Materials

   WT tobacco (N. tabacum “Hongda”) seeds were obtained from the National
   Infrastructure for Crop Germplasm Resources (Tobacco, Qingdao, China).
   Construction of the NtSPS1 OE tobacco plants was carried out as
   indicated below.

4.1.1. Construction of the NtSPS1 OE Vector

   NtSPS1 was cloned according to the sequence obtained from a BLAST
   search against the Gene-Space Sequence Reads from the China Tobacco
   Genome database, as described previously [[177]24]. Considering the
   restriction sites of the OE vector pCHF3 and the NtSPS1 fragment, the
   restriction site SmaI (CCCGGG) was added to the upstream primer for
   NtSPS1 and the restriction site SalI (GTCGAC) was added to the
   downstream primer for NtSPS1. The sequences of the primers with the
   adapters were 5′-TCCCCCGGGATGATGTCTGTGACTTGCCATAATCTTGAG-3′ (upstream
   primer) and 5′-CGCGTCGACCTATTCAATTCTCTCCAGATTATACTTCAC-3′ (downstream
   primer), and the Tm of the primers was 60 °C. The high-fidelity enzyme
   KD-Plus was used to amplify the target gene, and the products were
   subjected to agarose gel electrophoresis. Through a gel documentation
   system, the band corresponding to the size (~1200 bp) of the target
   fragment was subjected to gel extraction. The fragment was ligated to
   the cloning vector and transformed into competent Escherichia coli.
   Colonies containing the target fragments were selected via antibiotic
   resistance and PCR screening ([178]Figure 8A—most of the selected
   clones were positive) and were then verified by sequencing after
   propagation. Thus, the PCR-amplified NtSPS1 coding sequence was
   successfully ligated into the pCHF3 binary vector and cloned in E.
   coli.

Figure 8.

   [179]Figure 8
   [180]Open in a new tab

   PCR screening of Escherichia coli clones transformed with the
   pCHF3-NtSPS1 plasmid (A), agarose gel electrophoresis of NtSPS1 gene
   excised from a positive clone by SmaI/SalI double digestion (B), and
   PCR screening of bacterial clones transformed with NtSPS1 ligated into
   the OE vector (C). M—marker.

   Positive colonies with the expected sequencing results were further
   propagated, and positive plasmids were isolated from these colonies.
   The positive plasmids and OE vector were subjected to SmaI/SalI double
   digestion using the following digestion system at 37 °C for 2 h. The
   double-digested fragment of the NtSPS1 gene is shown in [181]Figure 8B.
   The digestion products from the positive plasmids and the OE vector
   were purified by agarose gel electrophoresis. The recovered products
   were ligated overnight at 16 °C, with a volume ratio of
   insert:vector:T4 DNA Ligase:T4 DNA ligase buffer of 5:3:1:1. The
   ligation mixture was subjected to transformation in E. coli. The PCR
   screening results of the NtSPS1 recombinants are shown in [182]Figure
   8C. The sequence-verified OE constructs obtained from the positive
   colonies were transformed into Agrobacterium tumefaciens for subsequent
   plant transformation.

4.1.2. A. tumefaciens–Mediated Genetic Transformation

   Fifty microliters of a stored A. tumefaciens culture was transferred to
   a 50 mL small conical flask containing LB medium with kanamycin and
   rifampicin. The flask was cultured in an incubator at 28 °C and 200 rpm
   constant shaking for approximately 12 h, until the color of the
   bacterial suspension turned from brownish red to dark yellow and its
   optical density (OD) reached approximately 0.6. The bacterial
   suspension was then transferred to a 50 mL centrifuge tube and
   centrifuged at 15,000× g for 10 min. After removing the supernatant,
   the bacterial pellet was resuspended in sterile water and, after
   adjusting the OD to approximately 0.6, acetosyringone was added (final
   concentration 20 mg/L). The bacterial suspension was then transferred
   to a large sterilized conical flask to prepare for the transfection.

   Tobacco leaves were transferred with tweezers from a tissue culture
   flask to sterilized filter paper. The edges and main veins of the
   leaves were removed with scissors, and the leaves were cut into 1.2 ×
   1.2 cm pieces. These small pieces were then transferred to the conical
   flask containing the bacterial suspension, shaken, and soaked for 8–10
   min. After this period, the small pieces were spread on co-cultivation
   medium, with their upper surfaces facing down. The culture dish was
   covered with plastic wrap, placed in a greenhouse, and cultured in the
   dark for 48–60 h. When the leaves started to differentiate, the
   differentiated shoots were excised, and transferred onto rooting
   medium. The rooting status of the seedlings was observed until the
   seedlings grew to ~5 cm in height, with 3–5 roots each. At this point,
   the lid of the tissue culture flask was unscrewed, an appropriate
   amount of sterile water was added, and seedling hardening was performed
   after cultivation in a greenhouse for approximately three days. The
   hardened seedlings were then transplanted to pots containing sterilized
   nutrient soil. After transplanting, water was provided as required to
   promote root system growth and the transgenic plants were later
   obtained.

4.1.3. Molecular Detection of the Transgenic Plants

   The transgenic-positive plants contained the Pchf3 vector with the 35S
   promoter, whereas the non-transgenic plants did not. Therefore, a
   forward primer annealing in the 35S promoter sequence and a reverse
   primer annealing in the NtSPS1 sequence (downstream primer
   5′-AAAGTCGACCTATTCAATTCTCTCCAGATTATACTTCAC-3′) were used for positive
   verification of the T0 NtSPS1 OE plants. The size of the amplified
   fragment was approximately 1400 bp. DNA from the transgenic tobacco
   leaves was used as template for PCR amplification. The plasmid DNA of
   the OE vector was used as the positive control, and DNA from
   non-transgenic tobacco leaves was used as the negative control. Agarose
   gel electrophoresis of the PCR products was performed to determine
   whether the transgenic plants were positive.

4.2. Plant Growth Conditions

   Seeds of the NtSPS1 OE and WT tobacco plants were germinated in a
   growth substrate that contained a mixture of vermiculite and peat (1:2,
   v/v). After germination, the plants (~15 cm height) were transferred to
   pots containing the above growth substrate. After eight weeks, the
   plants were moved to an illumination incubator (MGC-250; Yihengyiqi,
   Suzhou, Jiangsu, China) under day/night temperatures of 30/24 °C and a
   12-h photoperiod. After four weeks in the illumination incubator,
   tobacco leaves were sampled to determine photosynthetic gas exchange
   and chlorophyll fluorescence. Leaf samples were harvested from the
   fifth fully expanded leaf from the top of the plant at 0, 3, 6, 9, and
   12 DAS. The collected tobacco samples were mainly used for assessing
   solanesol and chlorophyll content and the metabolome.

4.3. Analysis of Total Solanesol Content

   Extraction of total solanesol from tobacco leaves of the NtSPS1 OE and
   WT tobacco plants was performed according to the procedure described by
   Yan et al. [[183]16]. The tobacco leaves were dried to constant weight
   using a freeze-dryer (Alpha 1–2 LD Plus; Christ, Osterode, Lower
   Saxony, Germany), ground, and sifted through a 40-mesh sieve. Portions
   (0.2 g) of the powdered samples were transferred to individual 20-mL
   centrifuge tubes with stoppers, and 1 mL 1 M NaOH (diluted in ethanol)
   and 5 mL hexane were added. Ultrasonic extraction was performed at 50
   °C for 30 min, and 8 mL distilled water was added. After centrifugation
   at 3000× g for 10 min, 0.5 mL of the supernatants was sampled, diluted
   with 4.5 mL methanol in brown volumetric flasks, and filtered through a
   0.2-μm membrane. Total solanesol content was measured using ultra-high
   performance liquid chromatography (ACQUITY UPLC H-Class; Waters,
   Milford, MA, USA) with an Atlantis T3-C[18] column (4.6 × 150 mm, 3 μm;
   Waters) that was maintained at 35 °C. A 50:50 (v/v)
   methanol-acetonitrile solution was used as the mobile phase at a flow
   rate of 1.0 mL/min, and a diode array detector was used for detection
   at 208 nm.

4.4. Quantitative Reverse Transcription PCR of NtSPS1

   Quantitative reverse transcription PCR (qRT-PCR) of NtSPS1 gene
   expression from the leaves of the NtSPS1 OE and WT tobacco plants was
   performed according to the procedure described by Yan et al. [[184]16].
   Gene-specific primer pairs were designed to detect NtSPS1 (upstream
   primer: 5′-CATTCCAAATATGAGATGCGTTGT-3′; downstream primer:
   5′-TGTGGACTTGGGAGAGGACT-3′). Two fragments of the constitutively
   expressed Ntactin gene were amplified as references using the