Overexpression of HvVDE gene improved light protection in transgenic tobacco (Nicotiana tabacum)

Plant materials and growth conditions

Plantlets of Hosta ventricosa Stearn were planted at Greenhouse A7 of Jilin Agricultural University Experimental Garden, in Jilin province, China. The cultivation conditions were an average temperature of 20.2°C, full sunlight, 60% humidity, and a photoperiod of 12/12 h, and the maximum light intensity of natural daylight was 3500 μmol m⁻² s⁻¹. When the H. ventricosa seedlings were 6 months old, we collected healthy root, stem, and leaf tissues, which were rapidly frozen in liquid nitrogen for subsequent experiments. Each tissue sample was sourced from at least three biological replicates. In additional, we conducted shading experiments using the aforementioned plants, which were randomly allocated into four groups, each consisting of 10 pots. Black nylon nets were employed to established shaded treatment, resulting in light transmission rates of 15% (ZQY), 30% (ZBG), and 50% (QGZ) in comparison to the non-shaded control group (CK), while maintaining consistent conditions for other growth parameters. The measured transmittance levels were determined using a ZDS-10 illuminance meter. Transmitance levels for the QGZ, ZBG, ZQY treatment groups were 55.38%, 34.12% and 16.41%. The maximum light intensities of shaded treatment groups were 1200 ± 10 μmol m⁻² s⁻¹ (QGZ), 600 ± 10 μmol m⁻² s⁻¹ (ZBG), and 100 ± 10 μmol m⁻² s⁻¹ (ZQY), respectively. Following a treatment duration of 7 days, leaf samples were collected from treatment groups and control groups, rapidly frozen using liquid nitrogen and stored at −80°C for further analysis.

The wild-type (WT) Nicotiana tabacum (L.) ‘NC89’ seedlings and transgenic tobacco lines were grown in a growth chambers maintained under controlled conditions. These conditions included a temperature of 25 ± 2°C, a humidity level of 70 ± 5%, and white light calibrated to mimic sunlight, with a light intensity of 100 ± 10 μmol m⁻² s⁻¹, and a photoperiod of 12 h of light and 12 h of darkness.

Cloning and bioinformation analysis of HvVDE

Total RNA was isolated utilizing the RNAprep Pure Extraction Kit (DP441, TIANGEN biochemical Technology, Beijing, China) and assessed for quality and integrity through 1.0% agarose gel electrophoresis. Subsequently, the initial complementary DNA (cDNA) strand was synthesized using the PrimeScript RT reagent Kit with gDNA (RR047, TaKaRa, Tokyo, Japan) following the manufacturer’s protocol. Specific primers were designed based on sequences derived from transcriptome data (Supplementary Table S1) using Permier Primer 6.0 software. The HvVDE gene was then amplified from the cDNA using TaKaRa Taq™ (R001A, TaKaRa, Tokyo, Japan) under the following cycling conditions: initial denaturation at 94°C for 3 min, followed by 33 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. Polymerase chain reaction (PCR) fragments were purified using a Gel Extraction Kit (CW2302 CWBIO, JiangSu, China), cloned into a pMD™ 18-T Vector Cloning Kit (6011 Takara, Tokyo, Japan), and sequenced by GENE Biotechnology company (Beijing, China). The conserved domain of the target gene was predicted using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/structure/cdd/wepsb.cgi). Alignment analysis of VDEs was conducted with DNAMAN, and a phylogenetic tree was constructed using MEGA 7.0 through the Neighbor-Joining method with 1000 bootstrap replicates.

Quantitative PCR (qPCR) analysis

The initial cDNA strand for qPCR was generated using the PrimeScript RT reagent Kit with gDNA Eraser (RR047, TaKaRa, Japan). The amplification process was conducted on a BioRad IQ5 instrument (Biorad, USA) utilizing the SYBR Prime Ex Tap II (RR420, TaKaRa, Japan) in accordance with the manufacturer’s instructions. The PCR protocol involved an initial denaturation step at 95°C for 30 s; followed by cycles of denaturation at 98°C for 5 s, annealing at 60°C for 34 s, for a total of 40 cycles; and a final extension step at 95°C for 15 s, annealing at 60°C for 60 s, and a final denaturation at 95°C for 15 s. The expression levels of HvVDE in various tissues and treatment groups of leaves were quantified using HvActin as the reference gene (Zhang et al. 2020).

The qPCR technique was utilized to identify the principal genes associated with photosynthesis and the xanthophyll cycle in tobacco. The experimental setup and procedures mirrored those described previously, with the primer sequences detailed in Table S1. The relative expression levels of the identified genes were assessed in comparison to a reference gene in the control group using the 2^−ΔΔCT method (Luo et al. 2020).

Construction of overexpression vector and Agrobacterium-mediated transformation

The fragment of HvVDE gene was introduced the Kpn I and BamH I restriction enzyme sites and was inserted into a pCAMBIA1300 vector to create the recombined vector pCAMBIA1300- HvVDE. Subsequently, the recombinant vector was transformed into the Agrobacterium EHA105 strain (Hwang et al. 2015), and preformed PCR to confirm the successful transformants. Then, the transformation of tobacco was performed using the leaf-plate method as previously described (Okuzaki and Tabei 2012). Transgenic lines displaying the highest expression levels of the HvVDE gene were identified through qPCR analysis and selected for further investigations. The growth status was observed and compared with the WT tobacco plants.

The subcellular localization of HvVDE

The full-length cDNA of HvVDE without stop codon were amplified and inserted into the BamH I site of the vector 16318hGFP. The fusion 16318hGFP-HvVDE vector was constructed and transformed into Arabidopsis protoplast. The empty 16318hGFP vector was also transformed as a control. Following the protocol described by Abel and Theologis (1994), 20-day-old leaves from Arabidopsis thaliana seedlings were chosen for the protoplast transformation procedure. The supernatant was subjected to enzymatic digestion, followed by treatment with W5 and MMG solutions to yield a protoplast suspension. Subsequently, the recombinant plasmid 16318h-GFP-HvVDE was introduced and incubated in darkness for a period of 2 days. The visualization of fluorescence signals within the protoplasts was achieved through examination using a laser confocal scanning microscope (Nikon C2-ER, Japan).

Determination of fluorescence characteristic parameters of transgenic tobacco

In a previous study conducted by Li et al. (2015), a portable chlorophyll fluorometer (FMS-2, UK) was utilized to assess the fluorescence parameters of healthy leaves of transgenic tobacco T1 lines under controlled light conditions, with consistent leaf positioning. Prior to the measurements, the leaves underwent a dark treatment for 15 min, followed by exposure to a low light intensity of 1 μmol m⁻² s⁻¹ to determine the initial fluorescence (F_o). Subsequently, a saturated pulsed light intensity of 1200 μmol m⁻² s⁻¹ was employed to measure maximum fluorescence (F_m), variable fluorescence (F_v = F_m – F_o), and maximum photochemical efficiency of Photosystem II (PSII) (F_v/F_m). Three leaves were selected for each experimental treatment.

To further analyze the components of NPQ, we employed a typical quenching analysis followed by relaxation analysis, applied to the dark-adapted, young and fully developed leaves from WT and overexpression (OE) lines using actinic light of 1200 μmol m⁻² s⁻¹. The time-course of NPQ in 25 min dark-adapted leaves was determined. F′m was defined as the maximal fluorescence during the illumination and F′m was defined as the maximal fluorescence during dark recovery after previous illumination, qE=Fm/F′m–Fm/F″m;qI=Fm/F″m–1.

Statistical analyses

Three distinct plants were utilized as individual replicates for each treatment. The data underwent a two-way analysis of variance (ANOVA), followed by the application of the Tukey test as a post hoc analysis. A significance level of P < 0.05 was established. Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA).

Isolation and characteristics of HvVDE

In order to investigate the gene function of HvVDE, gene-specific primers were designed based on contig sequences retrieved from a previous transcriptome database (Zhang et al. 2020). The open reading frame (ORF) sequence of HvVDE was successfully obtained through PCR amplification (Fig. 1a). A comparative analysis of the nucleotide sequence of the cloned gene with known homologous sequences of phytoene desaturase in different species was conducted using the NCBI database. The ORF with the length of 1446 bp, encoded a putative protein consisting of 481 amino acids with a molecular weight of 54.304 kDa and an isoelectric point (pI) of 5.19. The amino acid sequence of HvVDE exhibited the highest content of glutamate (Glu) and leucine (Leu) at 8.5%, followed by serine (Ser) at 7.7% and threonine (Thr) at 6.4%, but lacked pyrrolysine (Pyl) and selenocysteine (Sec). HvVDE was characterized as an unstable soluble protein with a hydrophobic index of −0.319 and a stability index of 47.25. Notably, HvVDE was found to lack a signal peptide, indicating its classification as a non-secretory protein.

Fig. 1.

Amino acid alignment and phylogenetic analysis of HvVDE and homologous proteins. (a) The PCR product of HvVDE. (b) The prediction of HvVDE protein structure. (c) Alignment of amino acid sequences of VDEs in different plants. Red lines indicate the conserved domain of the VDE protein. The pink and blue color means >75% and >50% homology among those sequences, respectively. (d) Phylogenetic tree of HvVDE constructed with MEGA 11.0, using the neighbor-joining (NJ) method and 1000 bootstrap replicates.

Moreover, the HvVDE protein was found to consist of 43.04% alpha helix, 16.63% extended strand, 6.65% beta-turn, and 33.68% random coils, and the protein structure of HvVDE is shown in Fig. 1b. Furthermore, examination of the conserved domain in the NCBI database revealed that HvVDE contains a PLN02372 conserved domain, which is categorized as a member of the violaxanthin de-epoxidase group within the lipid carrier protein superfamily.

Multi-alignment analysis and phylogenetic tree conduction

As shown in Fig.1c, multiple sequence alignment revealed that HvVDE predicted protein was highly similar to violaxanthin de-epoxidase proteins of other species (69.46–88.74%). Three regions with high homology for VDE were identified: a cysteine-rich region (I, E¹⁶²-F²⁰¹), a lipid transport protein signal structural region (II, P²⁹⁹-V³⁶⁰), and a high-charged glutamic acid-rich region (III, T³⁸⁰-V⁴¹⁵).

Furthermore, a phylogenetic analysis was conducted to explore the evolutionary relationship of HvVDE with other VDEs found in H. ventricosa and 11 additional plant species. Through a comparison of amino acid sequences, it was observed that HvVDE forms a closely related clade with JcVDE (XP_012092715.1) (Fig. 1d). This conservation of homology within the plant family highlights the crucial functional role of VDE.

Subcellular localization prediction analysis

To investigate the intracellular localization of the VDE protein, an analysis of the signal peptide of the HvVDE protein was conducted using SignalP 5.0. The results revealed the absence of a signal peptide sequence within the coding region of the HvVDE protein. Subsequent examination utilizing target P 2.0 software suggested a potential localization of this protein within the chloroplast.

To further validate the cellular localization of VDE, we generated HvVDE fusion constructs controlled by the CaMV35S promoter which were then expressed in Arabidopsis protoplasts. The results demonstrated that in protoplasts transfected with the empty control vector, the fluorescence signal was observed in both the nucleus and chloroplasts. Conversely, in protoplasts transfected with the recombinant vector, the fluorescence signal was exclusively localized in the chloroplasts. These results indicate that the protein encoded by the HvVDE gene is indeed situated within the chloroplasts, aligning with the anticipated localization (Fig. 2).

Fig. 2.

The subcellular localization of HvVDE by protoplast transformation. (a) Observation of GFP empty vector introduced in Arabidoposis protoplast. (b) Observation of HvVDE-GFP recombinant vector introduced in Arabidoposis protoplast.

Expression pattern of HvVDE

To study the expression profiles of HvVDE gene, qPCR analysis of HvVDE in tissues was performed. The HvVDE was modestly expressed in root and stem, but it expressed highly in leaf tissue (Fig. 3a). Furthermore, we detected the expression level of HvVDE under shading treatment groups. It is shown that the expression of the HvVDE gene was the highest in the QGZ group without shade, and the lowest in the ZQY group. This finding indicated that the expression of HvVDE gene is influenced by varying levels of light intensity.

Fig. 3.

The HvVDE transcript abundance in tissues and under the (a) light treatments and (b) light intensities determined by qPCR. Error bar, s.d. Lowecase letters represent significant differences between samples.

Besides, we further investigated the level of HvVDE transcription in H. ventricosa leaves exposed to different light intensities range from 300 to 1500 μmol m⁻² s⁻¹ for 2 h (Fig. 3b). The results showed that the level of HvVDE expression gradually increased with increasing light intensity, reaching the highest level at a light intensity of 1500 μmol m⁻² s⁻¹, which was 3.65 times higher than in complete darkness (0 μmol m⁻² s⁻¹).

Overexpression of HvVDE transgenic tobacco lines were generated by Agrobacterium-mediated transformation

Firstly, the Kpn I and BamH I were introduced to the open reading frame of HvVDE, and the recombined vector pCAMBIA1300-HvVDE-FLAG under the 35S promoter was conducted (Fig. 4a). Then, the freeze–thaw method was employed to transfer the recombined plasmid pCAMBIA1300-HvVDE to A. tumefaciens EHA105 and the positive transformants were confirmed through HvVDE specific PCR analysis (Fig. 4b). Through co-cultivation, the transgenic plantlets were regenerated, then the plantlets were screened on MS (Murashige and Skoog) medium plates supplemented with hygromycin (10 mg mL⁻¹). Resistant shoots were developed from the explants and transgenic plants of the T0 generation were obtained (Fig. 4d). The presence of HvVDE gene was validated through genomic DNA (gDNA) PCR using FLAG-Chek-F/R primers on leaves of T0 generation, where a 1400 bp band indicated successful introduction of HvVDE in transgenic tobacco (Fig. 4c). Plants transformed with an empty vector pCAMBIA1300 were used as controls. A total of 19 positive lines after Agrobacterium tumefaciens-mediated genetic transformation. The T0 tobacco seeds were germinated and cultivated on MS medium contained hygromycin (20 mg mL⁻¹) to obtain 15 genetically transgenic T1 tobacco lines harboring the HvVDE gene.

Fig. 4.

The construction of the overexpression vector and genetic transformation procedure. (a) The schematic of the HvVDE gene overexpression cassette. (b) Agarose gel showing the specific fragment for the HvVDE in recombinat plasmides; M, DL 2000 DNA marker; 1–4, the recombinat plasmide; 5, empty vector control. (c) The specific fragment for the HvVDE in transgenic tobacco line. (d) The process of Agrobacterium-mediated transformation in tobacco. (e) The relative expression level of HvVDE in the transgenic tobacco lines. Asterisks indicate that there is a significant difference between OE lines and WT; * P ≤ 0.05, ** P ≤ 0.01. Error bar, s.d.

qPCR was utilized to detect the expression of the HvVDE gene. The results showed that OE3, OE4, OE7, OE11, and OE15 exhibited high levels of HvVDE gene expression (Fig. 4e). Among these, OE3, OE4, and OE11 were selected for additional analysis due to their high expression levels. After selection we obtained T1 generation seeds of transgenic tobacco lines. Then, the T1 seeds of OE3, OE4, OE11, and WT tobacco were sown and grown under identical conditions. The results indicated no statistically significant differences in seed germination rates or plant height of the seedlings (Table S2). Furthermore, there were no observable differences in plant growth morphology or leaf color.

Photosynthesis determined in overexpression of HvVDE in tobacco

One of the most rapid and effective methods of protection involves the conversion of surplus absorbed light energy into heat energy. This can be accomplished by monitoring NPQ of chlorophyll fluorescence. To assess the impact of HvVDE heterologous expression on tobacco NPQ, we examined alterations in NPQ levels in transgenic tobacco and wild-type lines exposed to a light intensity of 1200 μmol m⁻² s⁻¹ (Fig. 5a). The findings indicate a progressive rise in non-photochemical quenching (NPQ) levels in both genetically modified tobacco and wild-type tobacco, with a notable surge observed within a 2-h timeframe. Throughout the duration of the treatment, the NPQ levels in genetically modified tobacco consistently exceeded those in wild-type tobacco. The finding suggests that under conditions of heightened light stress, genetically modified tobacco plants demonstrate a heightened capacity for energy dissipation through the xanthophyll cycle compared to their wild-type counterparts. The parameter F_v/F_m denotes the peak photochemical efficiency of the Photosystem II (PSII) reaction center in plants under optimal conditions, serving as an indicator of photoinhibition in the plant. Therefore, we measured the F_v/F_m of these genotypes and it showed that there is a notable decline in the maximum photochemical efficiency in conditions of intense light stress. The transgenic tobacco plants exhibit a greater F_v/F_m ratio in comparison to their wild-type counterparts (Fig. 5b).

Fig. 5.

(a) Non-photochemical quenching (NPQ) and (b) F_v/F_m levels in transgenic tobacco and WT. Asterisks indicate that there is a significant difference between OE lines with WT at test point; **P ≤ 0.01. Error bar, s.d.

The identification of the various components of NPQ was facilitated by their distinct relaxation times during a dark period following exposure to high light intensity. Fig. 6a illustrates the time-course of NPQ at light density of 1200 μmol m⁻² s⁻¹. Fig. 6b, c present the dark relaxation kinetics derived from Fig. 6a, specifically highlighting the rapid (qE) and slow relaxing (qI) components of NPQ. A substantial portion of NPQ was attributed to qE. In comparison to WT, OE lines exhibited a markedly higher qE and a lower qI.

Fig. 6.

NPQ relaxation analysis and calculation of energy quenching (qE) and photoinhibitory quenching (qI). (a) Time-course of NPQ in 25 min dark-adapted leaf. Black bar represents dark, white bar represents actinic light of 1200 μmol m⁻² s⁻¹. (b) qE and (c) qI were calculated. Vertical bars represent s.d. (n = 6). Asterisks denote significant differences between light treatments in WT and OE transgenic lines (**P < 0.01).