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Article

Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

by
Yanmei Shi
1,2,†,
Jinggong Guo
3,†,
Wei Zhang
4,†,
Lifeng Jin
2,
Pingping Liu
2,
Xia Chen
2,
Feng Li
2,
Pan Wei
2,
Zefeng Li
2,
Wenzheng Li
5,
Chunyang Wei
6,
Qingxia Zheng
2,
Qiansi Chen
2,
Jianfeng Zhang
2,
Fucheng Lin
2,
Lingbo Qu
1,
John Hugh Snyder
2 and
Ran Wang
2,*
1
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, Henan, China
2
National Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, Henan, China
3
Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng 475004, Henan, China
4
China National Tobacco Quality Supervision & Test Centre, Zhengzhou 450001, Henan, China
5
Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650031, Yunnan, China
6
Staff Training Academy of CNTC, Zhengzhou 450008, Henan, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2015, 16(12), 30438-30457; https://doi.org/10.3390/ijms161226243
Submission received: 20 October 2015 / Revised: 11 December 2015 / Accepted: 15 December 2015 / Published: 21 December 2015
(This article belongs to the Special Issue Plant Molecular Biology)
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Versions Notes

Abstract

:
Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Graphical Abstract

1. Introduction

Due to the ever increasing severity of environmental deterioration, both water scarcity and soil salinization have become major problems in agriculture that limit plant growth and cause serious economic losses [1]. Accordingly, the development of plants with high stress-tolerance traits is needed urgently [2,3]. An increasing number of studies have reinforced the assertion that the molecular manipulation of genes, such as those encoding antioxidant enzymes [4,5,6], transcription factors [7,8,9,10], and ion transporters [11,12], has the potential to overcome multiple limitations to agricultural productivity by creating stress-tolerant transgenic plants.
Carotenoids are terpenoids with a number of conjugated double bonds that contribute to their characteristic colors in the yellow to red range [13]. In plants, carotenoids play a critical role in the light absorption processes and protect the photosynthetic machinery from photo-oxidative damage by quenching triplet chlorophyll and singlet oxygen derived from excess light energy [14,15]. Additionally, carotenoids are precursors for the synthesis of the hormone abscisic acid (ABA), which functions in plants as an important signal in a variety of developmental processes and in adaptive stress responses to environmental stimuli [16,17]. Abiotic stresses can generate oxidative stress by increasing reactive oxygen species (ROS) production and/or by altering antioxidant defenses in plants [18]. Recently, many reports have demonstrated that increased carotenoid content in plants can improve tolerance to abiotic stresses such as high light conditions, UV irradiation, and salt stress, by scavenging ROS [19,20,21]. Moreover, carotenoid accumulation contributes to product quality and nutritional value for some crops, such as wheat [22], maize [23], tomatoes [24], potatoes [25], and watermelons [26].
The carotenoid biosynthetic pathway has been studied extensively in recent years [27]. Cyclization of lycopene by lycopene ε-cyclase (ε-LCY) and lycopene β-cyclase (β-LCY) is regarded as a key branching point in carotenogenesis in plants, as this is where the fate of lycopene shifts to the α-branch or the β-branch of the pathway, thereby determining the composition of the global carotenoid content. Over-expressing endogenous β-LCY in tomatoes caused a strong accumulation of β-carotene in the fruit that resulted from the near complete cyclization of lycopene [28]. Bang et al. found that a critical mutation in the red watermelon β-LCY allele might reduce β-LCY activity and thus result in the accumulation of lycopene [26]. Lutein accumulation is reduced or completely absent in the lut1 and lut2 mutants of Arabidopsis, owing to the lack of functional copies of the ε-carotene hydroxylase (ε-OHase) and lycopene ε-cyclase (ε-LCY) genes, respectively. These mutants also had increased accumulation of β-branch carotenoid compounds [29,30]. Transgenic tomatoes expressing β-LCY from citrus had increased β-carotene and total carotenoid content [31]. Chen et al. found that overexpression of the β-LCY gene in transgenic Arabidopsis enhanced plant tolerance to oxidative stress and salt stress [32]; these findings motivated us to investigate the function of Ntβ-LCY in carotenoid accumulation and in plant responses to drought and salt stress in tobacco.
Tobacco, a tetraploid plant species, has played a pioneering role in plant research, laying part of the groundwork for modern agricultural biotechnology. Modern tobacco cultivars have been developed to produce high carotenoid content, given that carotenoids are aromatic precursors for tobacco quality. CuiBi One (CB1, Nicotiana tabacum) is a famous tobacco cultivar in China, known for the high levels of carotenoids in its mature leaves. In this study, a lycopene β-cyclase gene named Ntβ-LCY1 from tobacco was chosen for cloning and function characterization. The transcript expression levels of the Ntβ-LCY gene were analyzed in different developmental stages and in response to salt, drought, and ABA treatment, using both RNA sequencing and quantitative real-time PCR (qRT-PCR). The function of the Ntβ-LCY1 gene in salt and drought stress tolerance was investigated with overexpression (OE) and RNA interference (RNAi) plants. Transgenic OE plants had significantly improved salt and drought tolerance compared to wild-type (WT) plants. Our results suggest that Ntβ-LCY1 plays an important role in carotenoid accumulation and tolerance to abiotic stress, and indicate that Ntβ-LCY1 may prove useful in potential applications for molecular breeding and/or biotechnology in plants.

2. Results

2.1. RNA Sequencing Analysis of Genes in the Carotenoid Biosynthetic Pathway and the Characterization of Ntβ-LCY Genes

To analyze the function of carotenoid biosynthetic genes in the CB1 cultivar, leaf samples were collected at the fast growing stage (FGS), the flowering stage (FS), the topping stage (TS), and the lower leaf maturity stage (LLMS). RNA sequencing was used to analyze the differential expression of various RNA transcripts. The transcript levels of the genes in the carotenoid biosynthetic pathway are displayed in Table 1. Interestingly, we found that two copies of β-LCY showed the highest transcript levels in the lower leaf maturity stage, although the transcript levels of most of the genes in the carotenoid biosynthetic pathway showed a declining trend from the flowering stage to lower leaf maturity stage, which suggested that the β-LCY gene might play an important role in the accumulation of carotenoids in mature tobacco leaves. There are two transcribed copies of β-LCY genes in the tobacco genome (China tobacco database V2.0). We found two β-LCY genes in the RNA sequencing results; these were designated as Ntβ-LCY1 and Ntβ-LCY2. It can be seen from the results of the RNA sequencing that Ntβ-LCY1 was more strongly expressed than Ntβ-LCY2 in all four of the tested developmental stages of tobacco, which suggests that Ntβ-LCY1 may be relatively more important for biological functions than Ntβ-LCY2. We designed primers to clone the coding region of Ntβ-LCY1 from a CB1 leaf cDNA library. The cloned gene was 1503 bp in length and was predicted to encode a 500 amino acid protein with a calculated MW (molecular weight) of 56.05 kDa and a predicted pI of 6.68. Subsequently, we used a similar approach to clone the full-length Ntβ-LCY1 gene from genomic DNA of CB1, and found that the complete gene sequence was 1503 bp in length, indicating that the Ntβ-LCY1 gene had no introns. Sequence alignment revealed that the putative protein encoded by Ntβ-LCY1 likely shared high sequence identity with Ntβ-LCY2 and six other known β-LCY protein sequences from Nicotiana tomentosiformis, Nicotiana sylvestris, Solanum tuberosum, Solanum lycopersicum, Capsicum annuum, and Arabidopsis (Figure 1, Table 2). The coding regions of the Ntβ-LCY1 and Ntβ-LCY2 genes were highly similar to each other, with 97.1% identity between the two nucleotide sequences and 96.8% identity between the two amino acid sequences. In addition, the sequence identity between Ntβ-LCY1 and β-LCY in Nicotiana tomentosiformis was 99.8%, while the sequence identity between Ntβ-LCY2 and β-LCY in Nicotiana sylvestris was 100%. The results were validated by phylogenetic analysis done in MEGA5 using the UPGMA method [33]. According to the phylogenetic tree, Ntβ-LCY1 was grouped with the β-LCY gene from Nicotiana tomentosiformis, while Ntβ-LCY2 was grouped with the β-LCY gene from Nicotiana sylvestris (Figure 2). The β-LCYs genes of Solanaceae plants were clustered into a separate branch.
Table
Table 1. Transcript levels of genes in the carotenoid biosynthetic pathway based on RNA sequencing analysis of samples from leaves of four different growth stages in tobacco, including the fast growing stage (FGS), the flowering stage (FS), the topping stage (TS), and the lower leaf maturity stage (LLMS). The full-length and coding sequences of gene in the carotenoid biosynthetic pathway are listed in Supplementary File 1.
Table 1. Transcript levels of genes in the carotenoid biosynthetic pathway based on RNA sequencing analysis of samples from leaves of four different growth stages in tobacco, including the fast growing stage (FGS), the flowering stage (FS), the topping stage (TS), and the lower leaf maturity stage (LLMS). The full-length and coding sequences of gene in the carotenoid biosynthetic pathway are listed in Supplementary File 1.
GeneTranscript IDGene IDSample_FGSSample_FSSample_TSSample_LLMS
PSY1mRNA_108630_cdsNtab052309050.0020.2626.2626.84
PSY2mRNA_24759_cdsNtab01410805.726.435.154.42
PSY3mRNA_28820_cdsNtab058261090.1931.835.0248.05
PSY4mRNA_3350_cdsNtab047014046.2935.5424.1726.29
PDS1mRNA_13725_cdsNtab074631079.5042.7456.1365.04
PDS2mRNA_73042_cdsNtab0595110103.6663.2280.4264.72
ZDSmRNA_101234_cdsNtab065384082.8962.5670.3962.62
CRTISO1mRNA_114973_cdsNtab063454044.6126.4233.4623.74
CRTISO2mRNA_122944_cdsNtab002730024.7914.2719.222.76
CRTISO3mRNA_78351_cdsNtab073608014.3415.2615.0318.19
β-LCY1mRNA_46713_cdsNtab026895054.0237.9737.5155.58
β-LCY2mRNA_18729_cdsNtab038339043.5634.129.1444.63
ε-LCY1mRNA_60735_cdsNtab000611048.6522.3129.7432.46
ε-LCY2mRNA_99724_cdsNtab045595026.7914.1311.9713.38
β-OHase1mRNA_106915_cdsNtab067792055.6326.4525.0830.60
β-OHase2mRNA_120276_cdsNtab08610904.221.652.480.16
β-OHase3mRNA_121754_cdsNtab048618077.5670.6973.0096.59
ε-OHase1mRNA_131608_cdsNtab029913028.1810.2123.9220.13
ε-OHase2mRNA_140553_cdsNtab089582027.0215.4519.1426.27
VDE1mRNA_114230_cdsNtab085842053.1439.8469.3221.80
VDE2mRNA_119637_cdsNtab0230700107.9984.8695.4541.86
VDE3mRNA_130498_cdsNtab07211108.825.5910.515.70
VDE4mRNA_86361_cdsNtab01890708.524.455.665.53
VDE5mRNA_95599_cdsNtab06071701.030.000.460.84
ZE1mRNA_119539_cdsNtab0136170316.73279.76267.74297.37
ZE2mRNA_42563_cdsNtab0384590381.6339.56352.04353.57
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Figure 1. Comparison of β-LCY amino acid sequences by Genedoc software from Nicotiana tabacum (1: Ntβ-LCY1; 2: Ntβ-LCY2; this work), Nicotiana tomentosiformis (3: Ntomε-LCY, XM_009618113.1), Nicotiana sylvestris (4: Nsyε-LCY, XM_009795141.1), Solanum tuberosum (5: Stε-LCY, XM_006351204.1), Solanum lycopersicum (6: Slε-LCY, XM_010313794.1), Capsicum annuum (7: Caβ-LCY, GU085267.1), and Arabidopsis thaliana (8: Atε-LCY, AF117256.1). Blank back ground, completely conserved region; Grey back ground, partly conserved region; White back ground, non-conserved region.
Figure 1. Comparison of β-LCY amino acid sequences by Genedoc software from Nicotiana tabacum (1: Ntβ-LCY1; 2: Ntβ-LCY2; this work), Nicotiana tomentosiformis (3: Ntomε-LCY, XM_009618113.1), Nicotiana sylvestris (4: Nsyε-LCY, XM_009795141.1), Solanum tuberosum (5: Stε-LCY, XM_006351204.1), Solanum lycopersicum (6: Slε-LCY, XM_010313794.1), Capsicum annuum (7: Caβ-LCY, GU085267.1), and Arabidopsis thaliana (8: Atε-LCY, AF117256.1). Blank back ground, completely conserved region; Grey back ground, partly conserved region; White back ground, non-conserved region.
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Figure 2. Phylogenetic analysis of the β-LCY genes in higher plants based on an alignment of the nucleotide sequences using MEGA5 software by the neighbor joining method. The bootstrap values were each estimated using 1000 replications.
Figure 2. Phylogenetic analysis of the β-LCY genes in higher plants based on an alignment of the nucleotide sequences using MEGA5 software by the neighbor joining method. The bootstrap values were each estimated using 1000 replications.
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Table
Table 2. Sequence identities of the deduced Ntβ-LCY proteins with those of other plants.
Table 2. Sequence identities of the deduced Ntβ-LCY proteins with those of other plants.
Amino Acid Ntomβ-LCYNsyβ-LCYStβ-LCYSlβ-LCYCaβ-LCYAtβ-LCY
Identity (%)Ntβ-LCY199.896.891.490.689.277.0
Ntβ-LCY297.010091.090.489.277.0

2.2. Examination of Ntβ-LCY Transcript Levels in Tobacco Organs and in Response to Stress Treatment

The spatial expression patterns of the Ntβ-LCY genes were investigated with qRT-PCR analysis of samples from different tissues (leaf, stem, root, and flower) at the flowering stage of Nicotiana tabacum grown under normal growth conditions. Ntβ-LCY1 and Ntβ-LCY2 were both strongly expressed in the leaves, had lower expression in stems and flowers, and were weakly expressed in roots (Figure 3A). The expression level of Ntβ-LCY1 in all organs was higher than those of Ntβ-LCY2, a result consistent with the results of the CB1 RNA sequencing analysis.
Ntβ-LCY expression was evaluated after salt stress, drought stress, and treatment with the plant hormone ABA with qRT-PCR (Figure 3B–D). There were two important trends in the Ntβ-LCY gene expression patterns following these stress treatments. First, the transcript levels of the Ntβ-LCY genes in tobacco leaves were upregulated in response to both salt and drought stress treatment (Figure 3B,C), and the expression of the Ntβ-LCY genes was strongly induced by ABA treatment (Figure 3D). Second, the expression of the Ntβ-LCY1 gene had a more pronounced response to stress treatment than the expression of Ntβ-LCY2. These results demonstrated that the Ntβ-LCY genes may take part in anti-stress processes in tobacco. Based on the differential degree of expression induction between the two genes, we speculate that Ntβ-LCY1 may play a relatively more significant role in the stress resistance processes of tobacco.

2.3. Characterization of Ntβ-LCY1 OE and RNAi Transgenic Tobacco Plants

Overexpressing and knockdown (RNAi) transgenic tobacco plants were generated to investigate the biological function of Ntβ-LCY1 in abiotic stress responses. A total of 26 Ntβ-LCY1 OE transgenic lines were obtained by hygromycin screening and PCR screening to amplify the inserted fragments spanning Ntβ-LCY1 and the Flag gene in the vector. There were no obvious phenotypic difference between the Ntβ-LCY1 OE and the WT lines (Figure 4A). Figure 4B shows that the PCR products of the six Ntβ-LCY1 OE lines have a 310 bp band corresponding to the size of Ntβ-LCY1-Flag product. Comparatively, there was no 310 bp band in the PCR products of WT lines. Based on qRT-PCR analysis of the Ntβ-LCY1 gene expression level (Figure 4C), six confirmed Ntβ-LCY1 OE transgenic T1 lines were chosen for further analysis.
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Figure 3. Ntβ-LCY expression. (A) Spatiotemporal expression of Ntβ-LCY in tobacco leaf, stem, root, and flower; (B,C) Relative expression levels of Ntβ-LCY following salt and drought stress, as compared with untreated control plants (control); (D) Relative expression level of Ntβ-LCY following ABA treatment. Error bars represent standard deviation (n = 3). The data presented here are representative of three independent experiments.
Figure 3. Ntβ-LCY expression. (A) Spatiotemporal expression of Ntβ-LCY in tobacco leaf, stem, root, and flower; (B,C) Relative expression levels of Ntβ-LCY following salt and drought stress, as compared with untreated control plants (control); (D) Relative expression level of Ntβ-LCY following ABA treatment. Error bars represent standard deviation (n = 3). The data presented here are representative of three independent experiments.
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Twenty-three independent Ntβ-LCY1 RNAi transgenic tobacco lines were verified by PCR and qRT-PCR to measure the Ntβ-LCY1 gene transcript level. Transgenic Ntβ-LCY1 RNAi lines exhibited abnormal phenotypes, including albino leaves and dwarfism, as compared to WT plants (Figure 4D), and some RNAi plants died during early developmental stages. Six T1 generation transgenic lines own with obvious phenotypes were selected for further analysis. Products of PCR using kanamycin-gene-specific primers from all six of the Ntβ-LCY1 RNAi lines had a 659 bp band (Figure 4E), and the transcript levels of Ntβ-LCY1 mRNA were significantly reduced in these RNAi plants (Figure 4F). Owing to the high degree of homology (97%), it was difficult to specifically silence only one copy. The expression of Ntβ-LCY2 was also inhibited in the RNAi plants, which had silencing efficiencies ranging from 51%–67% in the L1–L6 RNAi transgenic lines.
To further ascertain whether the Ntβ-LCY1 expression levels in tobacco were correlated with carotenoid accumulation levels, the mRNA expression level of various carotenoid biosynthetic pathway genes and the carotenoid content were measured in Ntβ-LCY1 OE and RNAi transgenic lines. The genes both up- and downstream of the Ntβ-LCY branch point in the carotenoid biosynthetic pathway, including phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene ε-cyclase (ε-LCY), β-carotene hydroxylase (β-OHase), zeaxanthin epoxidase (ZE), violaxanthin deepoxidase (VDE), neoxanthin synthase (NXS), were all expressed at significantly elevated levels (p < 0.05) in the leaves of the Ntβ-LCY1 OE lines as compared to the WT plants (Figure S1). Higher accumulation levels of carotenoids, including β-carotene, violaxanthin, neoxanthin, and lutein, as well as chlorophyll, were observed in the Ntβ-LCY1 OE lines as compared to the WT plants (Figure 5). Consistently, in the RNAi transgenic lines, all of the genes of the carotenoid biosynthetic pathway showed dramatically (p < 0.05) reduced transcript levels as compared with the WT line (Figures S2 and S3). As expected, the carotenoid and chlorophyll content was markedly decreased in the RNAi transgenic lines (Figure 6).
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Figure 4. Identification and characterization of expression of the transgenic Ntβ-LCY1 overexpression (OE) and RNAi plants by PCR and qRT-PCR. (A) Phenotypes of WT and Ntβ-LCY1 OE transgenic tobacco; (B) Confirmation of the presence of the Ntβ-LCY1 transgene construct in the OE transgenic plants based on PCR screening using primers flanking the Ntβ-LCY1 gene. Lane 1: Marker DL2000; Lane 2: positive control; Lane 3: negative control. Lanes 4–9 are six independently Ntβ-LCY1 OE transgenic lines; (C) Relative expression levels of Ntβ-LCY1 in OE transgenic plants; (D) Phenotypes of WT and Ntβ-LCY1 RNAi transgenic tobaccos; (E) Confirmation of the presence of the Ntβ-LCY1 transgene construct in the RNAi transgenic lines based on PCR screening using primers of the kanamycin gene, Lane 1: Marker DL2000; Lane 2: positive control; Lane 3: negative control; Lanes 4–9: six independently Ntβ-LCY1 RNAi transgenic lines; (F) Relative expression levels of Ntβ-LCY1 in RNAi transgenic tobaccos. M, Marker DL2000; P, positive control (using plasmid as the PCR template); WT, negative control (DNA from WT lines used as the PCR template); L1–L6, DNA from L1–L6 transgenic lines used as the PCR template. Error bars represent standard deviation (n = 3). The data presented here are representative of three independent experiments.
Figure 4. Identification and characterization of expression of the transgenic Ntβ-LCY1 overexpression (OE) and RNAi plants by PCR and qRT-PCR. (A) Phenotypes of WT and Ntβ-LCY1 OE transgenic tobacco; (B) Confirmation of the presence of the Ntβ-LCY1 transgene construct in the OE transgenic plants based on PCR screening using primers flanking the Ntβ-LCY1 gene. Lane 1: Marker DL2000; Lane 2: positive control; Lane 3: negative control. Lanes 4–9 are six independently Ntβ-LCY1 OE transgenic lines; (C) Relative expression levels of Ntβ-LCY1 in OE transgenic plants; (D) Phenotypes of WT and Ntβ-LCY1 RNAi transgenic tobaccos; (E) Confirmation of the presence of the Ntβ-LCY1 transgene construct in the RNAi transgenic lines based on PCR screening using primers of the kanamycin gene, Lane 1: Marker DL2000; Lane 2: positive control; Lane 3: negative control; Lanes 4–9: six independently Ntβ-LCY1 RNAi transgenic lines; (F) Relative expression levels of Ntβ-LCY1 in RNAi transgenic tobaccos. M, Marker DL2000; P, positive control (using plasmid as the PCR template); WT, negative control (DNA from WT lines used as the PCR template); L1–L6, DNA from L1–L6 transgenic lines used as the PCR template. Error bars represent standard deviation (n = 3). The data presented here are representative of three independent experiments.
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Figure 5. Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 5. Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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Figure 6. Carotenoid and chlorophyll content in control and Ntβ-LCY1 RNAi transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 6. Carotenoid and chlorophyll content in control and Ntβ-LCY1 RNAi transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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2.4. Functional Analysis of Ntβ-LCY1 under Salt Stress

To test whether overexpressing Ntβ-LCY1 in tobacco could enhance salt tolerance, six-week-old seedlings of WT and OE transgenic lines were treated with 300 mM NaCl. Under normal (no treatment) conditions, the increased Ntβ-LCY1 expression in the OE plants did not lead to any major observable effects in plant architecture or growth habit. When subjected to salt stress for three weeks, the three OE lines grew well, with slightly yellow leaves, whereas the leaves of the WT plants were severely wilted and chlorotic (Figure 7A). Analysis of the relative water content (RWC) showed that the RWC of the leaves from the OE plants was higher than that of WT leaves (Figure 7B). The carotenoid and chlorophyll content were also investigated in these plants treated with salt stress. Although there were significant reductions in carotenoid and chlorophyll content in both OE and WT lines following three weeks of salt treatment, the carotenoid content (β-carotene, violaxanthin, neoxanthin, and lutein) and the chlorophyll content was obviously higher in the leaves of the OE transgenic plants (Figure 7G).
Abiotic stress often results in the substantial accumulation of ROS, causing membrane damage in plants. Therefore, the accumulation of H2O2 and of superoxide radical anions (O2−) was evaluated in Ntβ-LCY1 OE transgenic plants grown under salt stress, using histochemical staining with 3,3,-diaminobenzidine (DAB, for H2O2) and nitro blue tetrazolium (NBT, for O2−). It can be seen from Figure 7C,D that leaves from OE lines exhibited less intense staining for both DAB and NBT than that of WT plants, indicating that OE plants accumulated lower levels of ROS under salt stress. Malondialdehyde (MDA) content is often assessed and used to represent the extent of lipid peroxidation and membrane injury in living cells [34,35]. Before salt treatment, there was no difference in the MDA content between the WT and the OE lines. However, following salt stress, the MDA content was significantly lower in the leaves of OE plants than in WT plants (Figure 7E). These results highlight the excellent salt stress resistance properties of the Ntβ-LCY1 OE transgenic plants. Given its known role as an important hormone in plant abiotic stress resistance, we also measured the ABA content in transgenic and WT plants. Under normal growth conditions, there were no significant differences in ABA content (p > 0.05) between the WT and OE lines. By contrast, following the salt stress treatment, ABA content was higher (p < 0.05) in the leaves of OE transgenic lines than in the leaves of the WT lines (Figure 7F).
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Figure 7. Effects of salt treatment on the T1 generation of Ntβ-LCY1 OE transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 OE plants after three weeks of treatment with 300 mM NaCl; (B) Relative water content in leaves of WT and OE plants after salt stress treatment; (C,D) 3,3,-diaminobenzidine (DAB) (C) and nitro blue tetrazolium (NBT) staining; (D) for evaluation the accumulation of H2O2 and O2− in WT and OE plants after salt stress treatment; (E,F) Malondialdehyde (MDA) and abscisic acid (ABA) content in the leaves of WT and OE plants, with or without salt stress treatment; (G) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic lines after three weeks of salt stress treatment. L1–L3, three lines of Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 7. Effects of salt treatment on the T1 generation of Ntβ-LCY1 OE transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 OE plants after three weeks of treatment with 300 mM NaCl; (B) Relative water content in leaves of WT and OE plants after salt stress treatment; (C,D) 3,3,-diaminobenzidine (DAB) (C) and nitro blue tetrazolium (NBT) staining; (D) for evaluation the accumulation of H2O2 and O2− in WT and OE plants after salt stress treatment; (E,F) Malondialdehyde (MDA) and abscisic acid (ABA) content in the leaves of WT and OE plants, with or without salt stress treatment; (G) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic lines after three weeks of salt stress treatment. L1–L3, three lines of Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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The Ntβ-LCY1 RNAi transgenic lines were also used to study plant responses to salt stress. Six week-old WT and RNAi transgenic plants were subjected to 300 mM NaCl stress treatment. It can be seen from Figure 8A that leaf wilting was more evident in the RNAi transgenic plants than in the WT plants after eight days of salt treatment. The RWC values of the RNAi plants were obviously lower than those of the WT plants after two weeks of salt stress treatment (Figure 8B). In addition, the MDA and the ABA content were also examined. As shown in Figure 8C,D, as compared to WT plants, the Ntβ-LCY1 RNAi transgenic plants had higher MDA content and lower ABA content before and after the salt treatment. The carotenoid and the chlorophyll content decreased dramatically in the RNAi transgenic plants as compared to the WT plants, following salt stress treatment (Figure 8E). These results indicated that the attenuated expression of the Ntβ-LCY1 gene in tobacco reduced plant tolerance to salt stress.
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Figure 8. Effects of salt stress on Ntβ-LCY1 RNAi transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 RNAi plants under salt stress for eight days; (B) Relative water content in WT and RNAi plant leaves after salt stress for two weeks; (C,D) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and RNAi plants with or without salt stress; (E) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 RNAi transgenic plants after two weeks of salt treatment. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 8. Effects of salt stress on Ntβ-LCY1 RNAi transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 RNAi plants under salt stress for eight days; (B) Relative water content in WT and RNAi plant leaves after salt stress for two weeks; (C,D) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and RNAi plants with or without salt stress; (E) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 RNAi transgenic plants after two weeks of salt treatment. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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2.5. Functional Analysis of Ntβ-LCY1 under Drought Stress

In order to investigate whether Ntβ-LCY1 is involved in the drought stress resistance of tobacco plants, seven-week-old WT and Ntβ-LCY1 OE transgenic plants from lines 4, 5, and 6 were used for drought stress assays. Under normal growth conditions, plants of the three OE transgenic lines showed no obvious abnormal morphological phenotypes as compared with WT plants. However, after three weeks of water deprivation, the Ntβ-LCY1 OE transgenic plants showed a reduced rate of leaf wilting (Figure 9A) and exhibited higher RWC values than did the WT plants (Figure 9B). The carotenoid content and chlorophyll content of the OE transgenic plants were higher than those of the WT plants following three weeks of drought treatment (Figure 9G). The histochemical staining assays indicated that the ROS content (H2O2 and O2−) (Figure 9C,D) and the MDA (Figure 9E) were lower in the Ntβ-LCY1 OE plants than in the WT plants. Additionally, following drought stress, the ABA content was significantly higher in the leaves of the OE plants than in the WT plants (p < 0.05) (Figure 9F).
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Figure 9. Effects of drought treatment on T1 generation Ntβ-LCY1 OE transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 OE plants after three weeks of drought stress; (B) Relative water content in leaves of WT and OE plants after drought stress; (C,D) 3,3,-diaminobenzidine (DAB) (C) and nitro blue tetrazolium (NBT) (D) staining for evaluation the accumulation of H2O2 and O2− in WT and OE plants after drought stress; (E,F) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and OE plants treated with or without drought stress; (G) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic plants after three weeks of drought treatment. L4–L6, three lines of Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 9. Effects of drought treatment on T1 generation Ntβ-LCY1 OE transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 OE plants after three weeks of drought stress; (B) Relative water content in leaves of WT and OE plants after drought stress; (C,D) 3,3,-diaminobenzidine (DAB) (C) and nitro blue tetrazolium (NBT) (D) staining for evaluation the accumulation of H2O2 and O2− in WT and OE plants after drought stress; (E,F) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and OE plants treated with or without drought stress; (G) Carotenoid and chlorophyll content in WT and Ntβ-LCY1 OE transgenic plants after three weeks of drought treatment. L4–L6, three lines of Ntβ-LCY1 OE transgenic plants. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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Following drought treatment for eight days, the leaves of Ntβ-LCY1 RNAi plants exhibited a more serious wilting phenotype than did the WT plants (Figure 10A). The RWC values (Figure 10B) and the ABA content (Figure 10D) decreased, while the MDA content (Figure 10C) increased in RNAi plants compared to the WT plants after two weeks of drought treatment. In addition, the carotenoid content and the chlorophyll content were significantly lower in the Ntβ-LCY1 RNAi transgenic plants than in the WT plants after drought treatment (Figure 10E).
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Figure 10. Effects of drought stress on Ntβ-LCY1 RNAi transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 RNAi plants under drought stress for eight days; (B) Relative water content in WT and RNAi plant leaves after drought treatment for two weeks; (C,D) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and RNAi plants with or without drought stress; (E) Carotenoids and chlorophyll content in WT and Ntβ-LCY1 RNAi transgenic plants after two weeks of drought treatment. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
Figure 10. Effects of drought stress on Ntβ-LCY1 RNAi transgenic plants. (A) Phenotypes of WT and Ntβ-LCY1 RNAi plants under drought stress for eight days; (B) Relative water content in WT and RNAi plant leaves after drought treatment for two weeks; (C,D) Malondialdehyde (MDA) and abscisic acid (ABA) content in leaves of WT and RNAi plants with or without drought stress; (E) Carotenoids and chlorophyll content in WT and Ntβ-LCY1 RNAi transgenic plants after two weeks of drought treatment. Error bars represent standard deviation (n = 6). The data presented here are representative of three independent experiments.
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3. Discussion

We used RNA sequencing data for the CB1 tobacco cultivar and the China tobacco database V2.0 to identify and investigate tobacco β-LCY genes expected to function in tobacco carotenoid biosynthesis. The function of the Ntβ-LCY1 gene was characterized in detail. As an allotraploid plant, tobacco has homologous copies of functional genes; there are two copies of Ntβ-LCY in tobacco, Ntβ-LCY1 and Ntβ-LCY2. Sequence analysis results showed that the Ntβ-LCY genes were highly conserved with homologous genes in other higher plants (Figure 1), suggesting that the tobacco Ntβ-LCY genes likely have similar biological functions to the β-LCY genes in other plants. Spatial-temporal expression analysis showed that Ntβ-LCY expression was, relatively, significantly higher in leaves than in other organs (Figure 3A), indicating that its functions were mainly in leaves. In all organs, Ntβ-LCY1 was expressed more strongly than Ntβ-LCY2. Characterization of Ntβ-LCY expression following treatment with salt and drought stresses indicated that Ntβ-LCY1 expression was more responsive to stress than was the expression of Ntβ-LCY2 (Figure 3B,C), implying that Ntβ-LCY1 likely has a relatively more important role in plant stress resistance than Ntβ-LCY2. ABA is known to act as an important signaling molecule in plant abiotic stress responses [36]. We found that the transcription levels of the Ntβ-LCY genes, especially that of Ntβ-LCY1, were dramatically upregulated in response to the exogenous application of ABA (Figure 3D), suggesting that these genes may possibly be involved in ABA-mediated stress responses.
Transgenic orchids with silenced PSY expression had lower carotenoid content than did WT plants, and had semi-dwarf and photo-bleaching phenotypes in plants. These changes were likely the result of unusual thylakoid membrane assembly or lipid phase changes of the membrane structure of the mutant plants [37]. Kim et al. [38] silenced the β-LCY gene in RNAi transgenic sweet potato calli; silencing significantly increased the total carotenoid content and led to a change in the color of transgenic calli from yellow to orange. The transgenic calli also enhanced the antioxidant activity compared to the nontransgenic (NT) calli. In the present study, OE and RNAi transgenic tobacco lines were generated for the Ntβ-LCY1 gene. Compared to WT plants, the Ntβ-LCY1 OE transgenic lines showed no morphological differences under normal conditions (Figure 4A). The Ntβ-LCY1 RNAi transgenic lines had obvious phenotypes, including bleached leaves and retarded growth (Figure 4D). Most of the T0 Ntβ-LCY1 RNAi transgenic seedlings died during the early growth stages, which indicated that the Ntβ-LCY1 gene plays a vital role in plant growth. Similar results were obtained by Pogson et al. [39]; genetic lesions in β-LCY were lethal in Arabidopsis. The carotenoid and chlorophyll content was dramatically decreased in the Ntβ-LCY1 RNAi transgenic lines that we generated (Figure 6), while the Ntβ-LCY1 OE transgenic lines had increased accumulation of carotenoids and chlorophyll (Figure 5). qRT-PCR results for the OE and RNAi lines helped to explain the variation in pigment composition that we observed in the transgenic plants. The fact that the regulation of Ntβ-LCY1 expression in tobacco had a strong impact on carotenoid content and on the expression levels of genes both up- and downstream of the Ntβ-LCY branch point of the pathway suggested that there might be a feedback mechanism in the regulation of the carotenoid pathway.
Salinity and drought are major abiotic environmental stressors, and plants can produce and accumulate numerous active oxygen species under stress conditions. The membrane stability of cells can be affected by lipid peroxidation caused by ROS [40]. Carotenoids provide protection for plants against oxidative stress as non-enzymatic antioxidants, by scavenging ROS generated due to excess excitation energy from chlorophyll during photosynthesis [41,42,43,44], and thus helping to maintain the redox state of the cell and facilitate proper functioning of the cell under stress. Over-expression of the PSY gene in transgenic Arabidopsis enhanced plant tolerance to reactive oxygen species under salt stress [21]. Silencing of the β-OHase genes in transgenic sweet potato resulted in elevated β-carotene and total carotenoid levels, as well as enhanced salt stress tolerance [45]. In a previous study, we found that silencing of the ε-LCY in Nicotiana benthamiana resulted in an increase in the accumulation of β-branch carotenoids and alleviated photoinhibition of Photosystem II in plants grown in low temperatures and under low light stress [46]. We further manipulated the Ntε-LCY expression levels in Nicotiana tabacum with transgenic technology and observed that strong accumulation of β-branch carotenoids and enhanced salt and drought tolerance resulted from the suppression of Ntε-LCY expression [47]. These results suggested that knocking down ε-LCY expression led to increased β-branch carotenoid biosynthesis and enhanced plant tolerance to environmental stresses. In the present study, we found that overexpressing the Ntβ-LCY1 gene in tobacco dramatically improved plant tolerance to salinity and drought, while silencing of the Ntβ-LCY1 gene decreased the ability of plants to tolerate salinity and drought stress. The ROS and MDA content were apparently lower in the Ntβ-LCY1 OE plants than in the WT plants (Figure 7C–E and Figure 9C–E), indicating that the extent of cellular membrane injury due to salt and drought stress was less severe in the transgenic plants than in the WT plants. We also observed from MDA analysis that more lipid peroxidation occurred in the RNAi plants than in the WT plants, not only under stress conditions but also under normal conditions (Figure 8C and Figure 10C), suggesting that the silencing of the Ntβ-LCY1 gene triggered severe oxidative damage and directly affected the normal growth of tobacco plants. The elevated accumulation of carotenoids improved the antioxidant activity of OE plants, while the lower carotenoid levels in the RNAi plants reduced the antioxidant capacity of plants. Transgenic sweet potato calli with silenced ε-LCY gene expression inhibited the production of H2O2 by increasing the carotenoid content in plants [48]. Transgenic Arabidopsis overexpressing β-LCY exhibited lower lipid peroxidation than WT plants, likely due to lower levels of MDA under abiotic stress conditions [32]. RWC is a typical phenotypic and physiological parameter used for evaluating plant vitality under stress conditions. Generally, water loss from plants largely depends upon stomatal aperture, which is closely regulated by ABA content [49,50]. The high RWC values in the Ntβ-LCY1 OE plants is likely related to increased ABA content in the leaves of these plants. ABA synthesis was reduced, as a downstream effect of the inhibition of the carotenoid metabolic pathway, by silencing of PSY in transgenic orchids [37]. Logically, it followed that the ABA content was lower in the Ntβ-LCY1 RNAi transgenic plants than in the WT plants in our study (Figure 8D and Figure 10D), implying that the sharp decline in carotenoid content obviously affected the downstream ABA biosynthesis. Downregulation of β-OHase and ε-LCY expression in sweet potato calli enhanced the accumulation of carotenoids and ABA, and further improved the tolerance to salt-mediated oxidative stress conditions [45,48]. Therefore, the increase in ABA content was more pronounced in the OE lines than in the WT plants under stress conditions (Figure 7F and Figure 9F), which might offer a reasonable explanation for the enhanced tolerance capacity to environmental stresses observed for the OE transgenic lines.

4. Materials and Methods

4.1. Plant Materials

Seeds of L. cv. Petit Havana SR1 (Nicotiana tabacum) were obtained from the stocks maintained in our laboratory. The L. cv. Petit Havana SR1 tobacco plants grew in a greenhouse maintaining day/night temperature at 28/23 °C and 16 h light photoperiod, at the National Tobacco Gene Research Center, Zhengzhou, China. Foliar discs (1.0 cm diameter) of L. cv. Petit Havana SR1 were excised from healthy and fully expanded tobacco leaves from six-week-old WT plants and used for plant transformation. Seeds of transgenic tobacco were planted on MS medium with 150 mg/L kanamycin (for RNAi plants) or 5 mg/L of hygromycin (for OE plants). Three weeks later, T1 tobacco seedlings with kanamycin (or hygromycin)-resistance were transferred into soil. The Nicotiana tabacum cultivar CB-1 plants used for cloning and for expression analysis were cultivated at the experimental farm in Yunnan Province, China. The tobacco leaves, stems, roots, and flowers at flowering stages used for the expression profiling were collected and stored at −80 °C. The effect of ABA (10 μM) on expression of Ntβ-LCY was tested by spraying a 10 μM ABA solution on six-week-old seedlings’ leaves and sampling the treated leaves 10 h later.

4.2. RNA Isolation and cDNA Preparation

An RNeasy Plant Mini Kit (Gene Answer, Beijing, China) was used to isolate total RNA of tobacco. For gene cloning and qRT-PCR analysis, first-strand cDNA was synthesized from total RNA using the Super Script First-Strand Synthesis System according to the manufacturer’s instructions (Takara, Japan).

4.3. cDNA Library Construction and Sequencing

For the synthesis of cDNA and Solexa sequencing, about 50 μg total RNA samples were prepared at concentrations of approximately 1000 ng/μL from tobacco leaves at four different growth stages: the fast growing stage, the flowering stage, the topping stage, and the lower leaf maturity stage. The cDNA library construction, sequencing, and bioinformatics analyses referred to the methods of Pang et al. [51].

4.4. Cloning of Ntβ-LCY1 and Vector Construction

The coding sequence of Ntβ-LCY1 gene was amplified by PCR using high-fidelity DNA polymerase (PrimeSTAR® HS DNA Polymerase, Takara, Otsu, Japan) from CB-1 leaves using the primers of β-LCY1-F & β-LCY1-R, and then cloned into the T vector (Takara). Clones containing the Ntβ-LCY1 gene were further sequenced to confirm their sequences.
To construct the vector for gene overexpression in transgenic tobacco, the coding sequence of Ntβ-LCY1 was amplified using the forward primer (β-LCY1-OE-F, including SpeI site) and the reverse primer (β-LCY1-OE-R, including KpnI site). The purified amplified gene fragment was then digested with SpeI and KpnI and ligated into the Sp1300-Flag plant vector (kindly provided by Professor Weiqiang Qian’s Lab at Peking University, Beijing, China).
For the construction of the Ntβ-LCY1 RNAi vector, primers were designed from the sequence of a partial CDS of Ntβ-LCY1 with attB sites using primers β-LCY1-RNAi-attB-F and β-LCY1-RNAi-attB-R. The partial Ntβ-LCY1 fragment used in RNAi study was amplified from the cloned Ntβ-LCY1 plasmid detailed above. Then the obtained PCR products were integrated into the RNAi expression vector (pHellsgate2, provided by Professor Weiqiang Qian of Peking University) by BP site-specific recombination (Invitrogen, Carlsbad, CA, USA). Primer sequences used in this study are listed in Table S1.

4.5. Plant Transformation and Confirmation

The construct of Ntβ-LCY1-pHellsgate2 and Ntβ-LCY1-Sp1300-Flag were introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterium-mediated leaf disc transformation was performed to generate transgenic tobacco [52]. WT plants were used as controls in the experiments. The transformed plants were screened on MS medium with cephalosporin (250 mg/L) and either kanamycin (150 mg/L, for Ntβ-LCY1-RNAi plants) or hygromycin (5 mg/L, for Ntβ-LCY1-OE plants) and the surviving seedlings were grown in a greenhouse to produce seeds following self-pollination. The transgenic T0 line seeds were screened by germination on MS media with kanamycin (150 mg/L, for Ntβ-LCY1-RNAi plants) or hygromycin (5 mg/L, for Ntβ-LCY1-OE plants). The obtained resistant plants were transplanted to a close greenhouse for use in further analyses. The transgenic plants were confirmed through PCR and qRT-PCR analyses. The genomic DNA was isolated from leaves of tobacco for the PCR experiments. The integration of Ntβ-LCY1 RNAi lines was confirmed by PCR using primers of nptII gene (nptII-F and nptII-R), while OE plants was confirmed with hygromycin gene primers (Hyg-F and Hyg-R) and Ntβ-LCY1 gene flanking primers (β-LCY1-flanking-F and Flag-R). Primer sequences used in this study are listed in Table S1.

4.6. Gene Expression Analysis

qRT-PCR analysis was used to analyze the relative expression levels of the Ntβ-LCY1 and other genes in the carotenoid biosynthetic pathway in transgenic and control plants. Briefly, the analysis was done with a Fluorescent Quantitative PCR Detector (Bio-Rad, Carlsbad, CA, USA) using SYBR Green fluorescence probe (Gene Answer, Beijing, China) and 26s RNA (or L25) internal reference gene. qRT-PCR amplification products were assessed by melting curve analysis and gel electrophoresis to ensure amplification specificity. Three technical replicates were evaluated for each biological sample. The thermal cycling program for qRT-PCR cycling was 95 °C for 3 min, and then 40 cycles of 95 °C for 20 s and 60 °C for 20 s. The relative expression level of each gene was calculated using the 2−ΔΔCt method [53]. The qRT-PCR primers of β-LCY1 and β-LCY2 were designed based on the 3’-untranslated region (UTR) sequences of Ntβ-LCY1 and Ntβ-LCY2, respectively. Primers used in the qRT-PCR analysis are listed in Table S2 [46].

4.7. Carotenoid and Chlorophyll Extraction and Quantification

Two hundred milligrams of freeze-dried leaf samples were used to extract carotenoids and chlorophyll with 25 mL of acetone. The samples were sonicated for 20 min and then centrifuged at 4 °C for 10 min at 6000 rpm. The obtained extract was filtered through a Millipore filter (0.22 μm, Shanghai Chuding Analytical Instruments Ltd., Shanghai, China) and analyzed by high performance liquid chromatography (HPLC, Agilent, Palo Alto, CA, USA).
For HPLC analysis, the carotenoids and chlorophyll were separated on an Agilent 1100 HPLC system with a C18 column (3.9 mm × 150 mm, 3 μm; Waters Corporation, Bedford, MA, USA) and analyzed with a diode array detector (DAD) at 448 and 428 nm. Solvent A was isopropanol. Solvent B was 80% acetonitrile-water.

4.8. Salt and Drought Stress Treatments

Six-week-old seedlings of Nicotiana tabacum cultivar CB-1 were subjected to salt and drought stress treatments. The mRNA expression of levels of the Ntβ-LCY genes in response to salt and drought stresses was examined. For the salt stress treatment, the seedlings were irrigated with 300 mM NaCl for 24 h. For the drought treatment, water was withheld from the plants for eight days.
Two-week-old transgenic and WT plants grown on MS medium were transplanted to pots filled with potting soil. The seedlings were cultivated for 4 weeks before salt and drought treatments. For the salt stress, plants were treated with 300 mM NaCl for three weeks for the OE lines or two weeks for the RNAi lines. For the drought treatment, water was withheld from plants for three weeks for the OE lines or two weeks for the RNAi lines. Phenotypic changes in the treated plants were carefully observed and photographed when obvious phenotypic appeared. The carotenoid and chlorophyll content, RWC, MDA, ABA, H2O2, and O2− were all measured (methods detailed below) following the stress treatments. Each treatment was repeated three times.

4.9. Relative Water Content (RWC)

The RWC of leaves in transgenic (OE and RNAi) and WT plants were analyzed after the salt and drought treatments. First, the fresh weight of equally-sized discs leaves from treated tobaccos was measured after excision (FW). The leaf discs were then soaked in water overnight and the weight of inflated leaves was measured (IW) after careful drying of excess water. The dry weight (DW) of completely drying discs was also weighted after drying at 80 °C for 48 h in an incubator. The following formula was used to calculate the RWC: RWC (%) = (FW − DW)/(IW − DW) ×100 [54].

4.10. Detection of H2O2 and O2−

Following the salt and drought stress treatments, H2O2 accumulation was analyzed with the 3, 3,-diaminobenzidine (DAB, Sigma, St. Louis, MO, USA) method, and O2− content was analyzed using nitroblue tetrazolium (NBT, Sigma) staining methods [55]. The leaf discs were excised from tobacco using a cork borer. These discs were then immersed in a solution containing 5 mg/mL DAB (pH 3.8) for 20 h or containing 0.5 mg/mL NBT for 20 h in the dark to analyze H2O2 and O2−, respectively. Stained samples were then depigmented in 75% (v/v) ethanol and 5% (v/v) glycerol at 80 °C for 10 min. After cooling to room temperature, the samples were transferred into fresh ethanol and finally photographed with a digital camera.

4.11. Determination of MDA Content

A thiobarbituric acid (TBA) method was employed to measure the content of Malondialdehyde (MDA) [56]. Three hundred milligram samples of tobacco leaves were ground in 5 mL of 5% (w/v) trichloroaceticic acid (TCA) and subsequently centrifuged for 10 min at 10,000 rpm. Then 2 mL of the resulting supernatant was transferred into a new tube and immediately mixed with 2 mL of TBA. The reaction mixture was heated at 98 °C for 30 min, cooled quickly on ice, and then centrifuged at 10,000 rpm for 20 min at 4 °C. Absorbance of the supernatant was measured at 450, 532, and 600 nm by an ultraviolet spectrophotometer (Cary100/300, Agilent). The MDA content, expressed as μmol/g, was calculated according to the following formula: MDA content (μmol/g) = C (μmol/L) ×V (L)/fresh weight (g) × 1000, where C (μmol/L) = 6.45× (A532 − A600) − 0.56A450 and V refers to the volume (L) of the extracting solution.

4.12. ABA Extraction and Quantification

Samples were prepared using a modification of the method reported by Liu et al. [57]. Tobacco leaves were ground to a powder in liquid nitrogen. One hundred milligrams of powder were dissolved into 1.0 mL of extraction solvent (CH3OH/H2O, 80/20, v/v, with internal standards ABA-d6 at 20 ng/mL). The mixture was sonicated for 30 min and then centrifuged at 4 °C for 5 min at 8000 rpm. The supernatant was then filtered through a 0.22-μm filter and evaluated with HPLC-MS/MS analysis.
A 1290 Infinity LC system coupled to a 6490 Triple Quad mass spectrometer (Agilent) was employed in the ABA determination. The chromatographic separation was completed using an Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 μm) held at 50 °C, with a sample injection volume of 5 μL. Mobile phase A (MA) was 0.001% formic acid in water and mobile phase B (MB) was acetonitrile. The flow rate was 200 μL/min. The gradient elution method and mass spectrometer instrumental parameters were the same as in [47].

4.13. Statistical Analysis

All data were expressed as the mean ± SD of three independent replicates. One-way ANOVA tests were performed with SPSS for Windows Version 16.0 (SPSS Inc., Chicago, IL, USA). Values of p <0.05 were considered to be statistically significant.

5. Conclusions

In conclusion, we functionally characterized a stress-responsive Ntβ-LCY1 gene from tobacco and confirmed its essential role in the survival of plants owing to its important role in carotenoid biosynthesis. Overexpressing of the Ntβ-LCY1 gene can improve drought and salt tolerance by enhancing the ROS scavenging capacity of tobacco, making it an important candidate for modulating responses to abiotic stress in tobacco and in other plants.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/16/12/26243/s1.

Acknowledgments

This work was supported by the Science Project of the Zhengzhou Tobacco Research Institute (902013CZ0620). We also thank He Xie (Yunnan Academy of Tobacco Agricultural Sciences, Kunming, Yunnan) and Nan Liu (China National Tobacco Quality Supervision & Test Centre, Zhengzhou, Henan) for their kind help in drought experiments, material collection, and revision of the manuscript.

Author Contributions

Yanmei Shi performed the experiments and wrote the paper. Jinggong Guo analyzed the RNA sequencing data and participated in writing the paper. Wei Zhang was responsible for CB1 sample collection. Lifeng Jin, Pingping Liu, Xia Chen, Feng Li, Pan Wei, Zefeng Li, Wenzheng Li, Chunyang Wei, Qingxia Zheng, Qiansi Chen, and Jianfeng Zhang contributed reagents, materials, and analysis tools to the experiments. Lingbo Qu and Fucheng Lin supervised the study. John Hugh Snyder revised the paper. Ran Wang designed and wrote the paper. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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MDPI and ACS Style

Shi, Y.; Guo, J.; Zhang, W.; Jin, L.; Liu, P.; Chen, X.; Li, F.; Wei, P.; Li, Z.; Li, W.; et al. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance. Int. J. Mol. Sci. 2015, 16, 30438-30457. https://doi.org/10.3390/ijms161226243

AMA Style

Shi Y, Guo J, Zhang W, Jin L, Liu P, Chen X, Li F, Wei P, Li Z, Li W, et al. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance. International Journal of Molecular Sciences. 2015; 16(12):30438-30457. https://doi.org/10.3390/ijms161226243

Chicago/Turabian Style

Shi, Yanmei, Jinggong Guo, Wei Zhang, Lifeng Jin, Pingping Liu, Xia Chen, Feng Li, Pan Wei, Zefeng Li, Wenzheng Li, and et al. 2015. "Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance" International Journal of Molecular Sciences 16, no. 12: 30438-30457. https://doi.org/10.3390/ijms161226243

APA Style

Shi, Y., Guo, J., Zhang, W., Jin, L., Liu, P., Chen, X., Li, F., Wei, P., Li, Z., Li, W., Wei, C., Zheng, Q., Chen, Q., Zhang, J., Lin, F., Qu, L., Snyder, J. H., & Wang, R. (2015). Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance. International Journal of Molecular Sciences, 16(12), 30438-30457. https://doi.org/10.3390/ijms161226243

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