Introduction

Protein synthesis in the cytoplasm begins with methionine, whereas protein synthesis in chloroplasts and mitochondria begins with N-formylmethionine. However, most mature proteins do not retain their initial N-terminal methionine (methionine in the cytoplasm and N-formylmethionine in the organelles), suggesting that N-formylmethionine is excised during the maturation of most proteins. N-terminal methionine excision is a ubiquitous protein modification1. This modification process in plants involves special mechanisms, and in addition to methionine aminopeptidase, peptide deformylase (PDF) has also been shown to be involved in the removal of N-formylmethionine2. PDF can catalyse the removal of formyl groups from the N-terminal formylmethionine of the primary polypeptide chain3 and has been shown to be widespread in eukaryotes2,4,5. In bacteria, the removal of N-terminal formyl groups is a prerequisite for subsequent N-terminal modifications, such as N-acetylation, N-methylation, and O-phosphorylation6,7,8.

Actinonin is a pseudopeptide with an N-terminal methionine analogue9 and the related N-formylhydroxylamine derivative BB-349710. This small molecule can bind very tightly to the active site of PDF, thus greatly weakening the deformation reaction11,12,13. Actinonin is the most potent PDF inhibitor found thus far, and it has been shown to specifically inhibit PDF in plants14 as well as bacteria9. Therefore, researchers have used actinonin to study the role of PDF in plant organelles. It has been shown that actinonin can reversibly bind to the active site of bacterial PDF with nanomolar affinity, resulting in the retention of formylmethionine on nascent proteins in bacterial cells13,15,16. Inhibition of PDF activity in bacterial cells with actinonin rapidly induces cellular stress responses, particularly those related to protein misfolding and membrane defects, followed by global downregulation of metabolic pathways17. These results suggest that PDF plays a crucial role in maintaining the normal life activities of bacteria.

Eukaryotic peptide deformylases are also sensitive to actinonin14,18. Exposure of Arabidopsis thaliana (A. thaliana) to actinonin resulted in leaf bleaching and severe growth retardation and had significant dose-dependent effects18. After high concentrations of actinonin were applied to pea leaves, the fresh weight and dry weight of the pea plants decreased significantly, and the leaves exhibited an albino phenotype18. Exposure of Chlamydomonas reinhardtii to actinonin led to rapid degradation of all the newly synthesized photosystem II (PSII) complex-associated proteins19. After actinonin treatment of tobacco plants, D1 protein accumulation in PSII components decreases, PSII decomposes, and eventually leads to leaf death20. Transmission electron microscopy revealed that the chloroplasts and mitochondria of a PDF1B-deficient rice mutant were seriously damaged and that the mitochondria were slightly altered21.

To date, PDF has been extensively studied only in prokaryotes and, to a lesser extent, in a few herbaceous plants, such as A. thaliana, rice, and tobacco. However, PDF genes in woody plants have not been studied. Eucommia ulmoides Oliv (E. ulmoides) is not only an important traditional medicinal plant in China but also a high-quality gum source plant. With the exploration of its medicinal and gum value, this plant has received increasing attention from researchers. In this study, we isolated the EuPDF1B promoter and gene from E. ulmoides and characterized its specificity and induced expression pattern. Moreover, its function was analysed in transgenic tobacco overexpression lines.

Results

Isolation and identification of the EuPDF1B

The full-length sequence of EuPDF1B is 1176 bp in length with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids (Fig. 1). The CD-search tool in NCBI was used to predict the conserved domains of EuPDF1B, and the results showed that the protein has three absolutely conserved and unique motifs, which together constitute the entire active site of PDF. The amino acid sequence of this protein was compared with PDF amino acid sequences of other species, including Escherichia Coli (E. Coli) (accession no. CAA54826.1), A. thaliana (accession no. AF250959.1 and AF269165.1) and Solanum lycopersicum (S. lycopersicum) (accession no. AF250958.1 and AF271258.1), and an evolutionary tree was constructed using the neighbour-joining method in MEGA version 2.1. The results showed that the protein can be grouped with AtPDF1B and LePDF1B in the phylogenetic tree (Fig. 2). The above information confirmed that the cloned gene was indeed PDF1B and was named EuPDF1B.

Figure 1
figure 1

Nucleotide and deduced amino acid sequence of the EuPDF1B from E. ulmoides. The underlined section in front of the ATG indicates the 5’ end untranslated regions. The underlined portion after TGA indicates the 3’ end untranslated regions.

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Figure 2
figure 2

Phylogenetic tree of PDFs from E. coli, A. thaliana (AtPDF1A and AtPDF1B), S. lycopersicum (LePDF1A and LePDF1B) and E. ulmoides (EuPDF1B). The scale corresponds to 10 amino acid residues.

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Expression patterns of EuPDF1B in E. ulmoides

The expression level of EuPDF1B in different tissues of E. ulmoides seedlings and male and female plants was analysed by qRT‒PCR. The results showed that the expression of EuPDF1B in the leaves was significantly greater than that in the other tissues. In the seedlings, the expression level of EuPDF1B in the leaves was 3 times that in the roots and 6 times that in the stems (Fig. 3a). In male and female plants, the expression level of EuPDF1B in mature leaves was also significantly greater than that in other parts (Fig. 3b). This finding is consistent with the finding that the expression of PDF1B in mature leaves of rice is greater than that in young leaves22. Since leaves are the main organs involved in photosynthesis in plants and EuPDF1B is expressed mainly in leaves, it is speculated that EuPDF1B may play an important role in maintaining normal photosynthesis in plants.

Figure 3
figure 3

Expression patterns of EuPDF1B in E. ulmoides. a Relative expression levels of EuPDF1B in E. ulmoides seedlings. b Relative expression levels of EuPDF1B in E. ulmoides male and female plants. The error bars represent standard deviations of three replicates, ***P < 0.001, ****P < 0.0001.

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Subcellular localization analysis of EuPDF1B

Subcellular localization revealed that the positions of chloroplast autofluorescence and green fluorescence from EuPDF1B-GFP overlapped, while the positions of green fluorescence and red fluorescence did not overlap (Fig. 4). These results indicate that EuPDF1B is located in chloroplasts but not in mitochondria, in contrast to the finding that PDF1B in rice and S. lycopersicum is located in both chloroplasts and mitochondria22; the specific reasons for this difference need to be further studied. EuPDF1B is located in chloroplasts, which is consistent with the finding that the expression level of EuPDF1B in leaves is significantly greater than that in other parts, suggesting that EuPDF1B may play an important role in the maintenance of plant photosynthesis.

Figure 4
figure 4

Subcellular localization of EuPDF1B. Scale bars = 10 μm.

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Overexpression of EuPDF1B promotes tobacco plant growth

Transgenic tobacco plants overexpressing EuPDF1B were identified by GUS staining and PCR and three plant lines (TP1, TP2, TP3) with high expression level of EuPDF1B were selected for subsequent experimental study. Compared with those of wild-type tobacco plants and transgenic tobacco plants cultured under the same conditions, the average plant height, fresh weight and net photosynthetic rate of the transgenic tobacco plants were significantly greater than those of wild-type tobacco plants, but there was no significant difference in total chlorophyll content (Fig. 5a–c). These results indicated that the overexpression of EuPDF1B promoted the growth of tobacco plants. Previous studies have reported that silencing PDF1B can affect the expression of chloroplast coding genes. Considering that the D1 protein in PSII is the preferred substrate for PDF1B deformylation23 and is also encoded by chloroplast genes, we examined the expression of the psbA gene, which encodes the D1 protein, in transgenic tobacco. The expression level of psbA in the transgenic tobacco plants was 4 times greater than that in the wild-type tobacco plants (Fig. 5d), indicating that the overexpression of EuPDF1B promoted the expression of psbA in tobacco plants. Moreover, the average number of chloroplasts in each mesophyll cell of the transgenic tobacco plants was 4 more than that in the wild-type tobacco plants, but the difference was not significant. However, the chloroplast grana thylakoid membranes of the transgenic tobacco plants were more densely arranged than those of the wild-type tobacco plants (Fig. 5e).

Figure 5
figure 5

Characterization of the tobacco EuPDF1B line. a One-month-old wild-type and EuPDF1B transgenic plants. b Phenotypic indicators. c Physiological indexes. d Relative expression levels of genes. e TEM observation of chloroplasts. WT: wild-type tobacco plants, TP: transgenic tobacco plants. The error bars represent standard deviations of three replicates, ****P < 0.0001. Cp indicates chloroplasts.

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Isolation and cis-element analysis of the EuPDF1B promoter

In this study, we cloned the 1714 bp promoter region of the EuPDF1B gene. The PlantCARE online website was used to analyse and predict the cis-acting elements in the promoter sequence. The results showed that the promoter region contained core promoter elements such as the CAAT box and TATA boxes as well as cis-acting elements related to the light response and hormone response, including 1 GT1 motif, 2 G-boxes, 1 GATA motif, 1 CTT motif, 1 GARE motif, 3 CGTCA motifs and 3 ABREs. The important cis-acting elements in the EuPDF1B gene promoter are listed in Table 1.

Table 1 Putative cis-acting regulatory elements identified in the promoter region of EuPDF1B.
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Analysis of EuPDF1B promoter activity

To determine the activity of the EuPDF1B promoter, four plant expression vectors with the GUS reporter gene driven by the EuPDF1B promoter with different lengths and missing different response elements were constructed (Fig. 6a). Finally, transgenic tobacco plants were generated by incubating the leaf discs with the genetically engineered Agrobacterium. Histochemical GUS staining was performed on these transgenic tobacco plants, and the results showed that the wild-type tobacco plants did not stain blue, but all transgenic tobacco plants (EuPDF1Bp-1, EuPDF1Bp-2, EuPDF1Bp-3, EuPDF1Bp-4) stained blue (Fig. 7), indicating that promoters of all four lengths drove GUS reporter gene expression; that is, the core promoter of the EuPDF1B promoter was located in the − 236 bp region. The roots, stems and leaves of the transgenic tobacco plants were stained blue, but the blue colour in the leaves was deeper (Fig. 7), indicating that the EuPDF1B promoter mainly drives the expression of downstream genes in the leaves. The blue colour of EuPDF1Bp-3 and EuPDF1Bp-2 were relatively dark, and the blue colour of EuPDF1Bp-1 and EuPDF1Bp-4 were relatively light (Fig. 7), indicating that there may be enhancer elements in the − 891 bp to − 236 bp region of the EuPDF1B promoter. These results were confirmed by qRT‒PCR (Fig. 6b).

Figure 6
figure 6

Analysis of EuPDF1B promoter activity. a Construction of GUS gene expression vectors driven by the EuPDF1B promoter with different length. b Relative expression levels of GUS driven by the EuPDF1B promoter with different length. The error bars represent standard deviations of three replicates, ***P < 0.001, ****P < 0.0001.

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Figure 7
figure 7

GUS protein expression in transgenic tobacco plants. Representative images showing GUS immunostaining of young shoots, roots, stems, leaves and buds of wild-type plants (WT), EuPDF1Bp-1, EuPDF1Bp-2, EuPDF1Bp-3, and EuPDF1Bp-4 transgenic plants. Scale bar = 3 mm.

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Expression profiles of EuPDF1B under shading and hormone treatments

Sequence analysis of the EuPDF1B promoter revealed photoresponsive elements, methyl jasmonate (MeJA) response elements, gibberellin (GA) response elements and abscisic acid (ABA) response elements. To verify whether light, MeJA, GA and ABA had any effect on the expression of EuPDF1B in E. ulmoides seedlings, qRT‒PCR was used to measure the expression of EuPDF1B after GA3, ABA, MeJA and shading treatment. After ABA or MeJA treatment, the expression of EuPDF1B gradually increased, peaked at 12 h after treatment and then rapidly decreased to a lower level than that before treatment (Fig. 8). After GA3 treatment, the expression level of EuPDF1B increased slightly and was 1.5 times that before treatment at 24 h after treatment (Fig. 8). After 2 days of shade treatment, the expression level of EuPDF1B decreased and was only 0.5 times that of the control plants (Fig. 8). These results suggest that the expression of EuPDF1B is regulated by GA3, ABA, MeJA and light.

Figure 8
figure 8

Relative expression levels of EuPDF1B in E. ulmoides seedlings under hormone and shading treatment. CK: negative control. The error bars represent standard deviations of three replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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To further determine whether light, MeJA, GA and ABA affect the expression of the EuPDF1B gene by affecting the activity of the EuPDF1B gene promoter, qRT‒PCR was used to determine the expression of GUS in the EuPDF1Bp-1, EuPDF1Bp-2, EuPDF1Bp-3 and EuPDF1Bp-4 transgenic tobacco plants after GA3, ABA, MeJA and shading treatment. GA3 induced GUS expression in the EuPDF1Bp-4, EuPDF1Bp-3, EuPDF1Bp-2 and EuPDF1Bp-1 plants. GA3, ABA and MeJA induced GUS expression in EuPDF1Bp-3, EuPDF1Bp-2 and EuPDF1Bp-1 plants, but the degree of GUS induction in EuPDF1Bp-2 was not as strong as that in EuPDF1Bp-1 and EuPDF1Bp-3. The expression level of GUS was increased in EuPDF1Bp-1 and EuPDF1Bp-4, decreased in EuPDF1Bp-2, and unchanged in EuPDF1Bp-3 after shading treatment (Fig. 9). These results were consistent with the predicted results of the promoter region, suggesting that GA, ABA and MeJA can promote the expression of EuPDF1B by increasing the activity of the EuPDF1B promoter, while low light can inhibit the expression of EuPDF1B by decreasing the activity of the EuPDF1B promoter.

Figure 9
figure 9

Relative expression of GUS under hormone and shading treatment. CK: negative control. The error bars represent standard deviations of three replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Discussion

The biosynthesis of all proteins begins with methionine. In the cytoplasm of eukaryotes, protein synthesis begins only with methionine, whereas in prokaryotes, as well as in the mitochondria and chloroplasts of eukaryotes, protein synthesis begins with N-formylmethionine24. The process of removing formyl groups from nascent peptide chains appears to be a conserved pathway in protein maturation. A mitochondrion-targeted PDF (PDF1A) and a chloroplast- and mitochondrion-targeted PDF (PDF1B) in higher and lower plants have been shown to be involved in the removal of the protein N-terminal formylmethionine in organelles2,4.

The retention of N-terminal formylmethionine in chloroplast-encoded proteins did not affect the accumulation of plastid RNA polymerase or plastid ribosome components but limited the accumulation of one or several PSII components and affected chloroplast PSII function19. Further studies have shown that treating tobacco seedlings with actinonin results in a rapid reduction in the accumulation of nascent D1 protein, which ultimately leads to the disintegration of the PSII complex, loss of PSII system activity, and leaf death20. Crystal structure and gel filtration chromatography evidence suggest that PDF1B is required in plants and has preferred substrate specificity for D1 polypeptides in PSII23. PSII in plant chloroplasts is essential for photosynthesis, accomplishing water photolysis and plastoquinone reduction by capturing light energy. As the photoreactive central protein of PSII, the D1 protein encoded by the chloroplast gene psbA plays a crucial role in the biogenesis and functional maintenance of PSII. Photosynthetic organisms encounter various abiotic stresses, such as low temperature, high temperature, and strong light, in their growth environment. The PSII complex of plants is the most vulnerable to damage from various abiotic stress processes25,26, but plants have evolved a repair process to renew damaged PSII complexes. This repair process involves the degradation and resynthesis of damaged proteins, particularly D1 proteins encoded by the chloroplast gene psbA, as D1 proteins are particularly vulnerable to damage during various abiotic stress processes27. Therefore, the rate of synthesis and degradation (or turnover) of D1 proteins represents a critical part of the PSII repair cycle. This cycle places the PSII complex in a photochemically active state28. Degradation of the damaged D1 protein begins with exposure to the N-terminus of the chloroplast matrix. Retention of the N-terminus formyl group of the D1 protein prevents the FtsH complex from recognizing the N-terminus of the D1 protein, thereby affecting the degradation of the D1 protein by the FtsH complex29. Blockage of D1 protein degradation affects the renewal and repair of damaged PSII. Since the degradation of the D1 protein during repair in PSII depends on the correct processing of its N-terminus, the removal of the N-terminal formylase by peptide deformylase is particularly important.

In transgenic tobacco plants overexpressing the EuPDF1B gene, the higher the expression level of EuPDF1B, the greater the expression level of psbA, indicating that the expression of EuPDF1B promotes the expression of the psbA gene. This finding is reasonable because the D1 protein encoded by psbA is the preferred substrate for the protein encoded by EuPDF1B. In addition, the height, fresh weight and net photosynthetic rate of the transgenic tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants. This may be because the overexpression of EuPDF1B promotes the expression of the psbA gene, which produces more D1 protein to participate in the assembly of PSII and timely replacement of the D1 protein damaged under abiotic stress, thus promoting the growth of tobacco plants. This finding is consistent with the observation that the chloroplast thylakoid membranes of transgenic tobacco plants overexpressing the EuPDF1B gene are more neatly arranged and densely packed than those of the wild type.

Moreover, it has been reported that the PDF1B gene of A. thaliana is involved in stress responses1. Therefore, it is speculated that PDF may play an active role in the process of plant resistance to abiotic stress. In our study, EuPDF1B expression was significantly induced by MeJA and ABA treatments. In addition, cis-acting element analysis of the EuPDF1B promoter revealed that it contains three ABA responsive elements (ABREs) and three MeJA responsive elements (CGTCA motifs), which respond to abiotic stress signals. This finding suggests that EuPDF1B may be involved in resistance to abiotic stress.

Histochemical GUS staining indicated that the EuPDF1B promoter mainly drives GUS gene expression in leaves. This result is consistent with the finding that EuPDF1B is expressed mainly in the leaves of E. ulmoides. In rice, the expression level of PDF1B in mature leaves was the highest among all tissues22. These results indicate that the expression patterns of PDF1B are similar in various plants. In this study, EuPDF1B was localized in chloroplasts but not in mitochondria, in contrast to the finding that PDF1B in rice and S. lycopersicum is located in both chloroplasts and mitochondria22. EuPDF1B is derived from the genome of E. ulmoides. There are differences in organ structure between woody plants and herbaceous plants, and the upstream regulatory sequences of the two plants are different, so the location of PDF1B may also be different. In addition, the expression level of GUS in EuPDF1Bp-3 and EuPDF1Bp-2 transgenic plants was significantly greater than that in EuPDF1Bp-1 and EuPDF1Bp-4 plants, indicating that enhancer elements were likely to be present in the − 891 bp ~ − 236 bp region of the EuPDF1B promoter. In our study, we identified and functionally characterized EuPDF1B, which laid a foundation for further analysis and study of PDF in woody plants.

Materials and methods

statement

None of the plants used in this study were wild and all the methods were complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Plant materials, growing conditions and treatment methods

Mature seeds of E. ulmoides were collected from the Agricultural Biotechnology Base of Guizhou University. Wild-type tobacco seeds were preserved in laboratories. Both the male and female E. ulmoides plants used in the experiment were planted at the Agricultural Biotechnology Base of Guizhou University. Both the E. ulmoides seedlings and tobacco plants were grown under the following conditions: 16 h light/8 h dark photoperiod at 25 °C and 50% relative humidity. Fully developed tobacco leaves were used for genetic transformation tests. Leaves of 2-months-old E. ulmoides seedlings or transgenic tobacco plants were sprayed with 100 µM MeJA, 100 µM ABA or 100 µM GA3, and control plants were sprayed with an equal volume of solvent. The leaves were collected at 0, 3, 6, 12, 24 and 48 h after spraying. Leaves of 2-months-old E. ulmoides seedlings or transgenic tobacco plants were collected after 50% shade treatment for 2 days. All the samples were frozen in liquid nitrogen after collection and stored at − 80 °C until RNA isolation. qRT‒PCR was used to determine the expression level of EuPDF1B in E. ulmoides and the GUS reporter gene in tobacco plants.

Cloning and sequence analysis of EuPDF1B and its promoter

After total RNA was extracted from E. ulmoides, reverse transcription reactions and 3′- and 5′-end amplification of the EuPDF1B gene were performed using a SMARTer RACE 5′/3′ kit (TaKaRa, Dalian, China). By sequencing and splicing the 3′ and 5′ ends of the EuPDF1B gene, a maximum ORF of 831 bp was obtained. Then, EuPDF1B was amplified by PCR, and the amplified product was constructed with the pMD18-T vector. Total DNA was extracted from the leaves using the CTAB method30. Then, specific primers were designed to amplify EuPDF1B promoter sequences of different lengths, and the cloned promoter fragments were constructed with the pMD18-T vector. The constructed vectors were subsequently sent to BGI for sequencing. The primers used are shown in Table 2. The CD-search tool in NCBI was used to predict the conserved domain of EuPDF1B, and the EuPDF1B promoter sequence of E. ulmoides was analysed by bioinformatics using the PlantCARE online website to identify possible regulatory motifs.

Table 2 Primers used for PCR.
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Construction of the plant transformation vector and genetic transformation of tobacco

The maximum open reading frame sequence of EuPDF1B was inserted into the plant binary vector pCAMBIA1301 under the control of the CaMV35S promoter by the double enzyme digestion method to obtain the overexpression vector pCAMBIA1301-35S: EuPDF1B. EuPDF1B promoter DNA fragments of different lengths (1714 bp/1308 bp/891 bp/236 bp) were connected to the EcoRI and BamHI enzyme restriction sites of the pCAMBIA1381Z vector to obtain the following four vectors: pCAMBIA1381Z-EuPDF1Bp-1: GUS (1714 bp); pCAMBIA1381Z-EuPDF1Bp-2: GUS (1308 bp); pCAMBIA1381Z-EuPDF1Bp-3: GUS (891 bp); pCAMBIA1381Z-EuPDF1Bp-4: GUS (236 bp). These vectors were used to transform Agrobacterium GV3101, which was subsequently genetically transformed into tobacco. PCR was used to identify the transgenic tobacco plants for further study. The primers used are shown in Table 2.

Subcellular localization analysis of EuPDF1B

The maximum open reading frame sequence of EuPDF1B without a stop codon was inserted into the pCAMBIA1300-GFP vector to obtain the fusion expression vector pCAMBIA1300-35S: EuPDF1B-GFP. Moreover, the pCAMBIA1300-35S: COX-RFP vector was constructed to determine whether EuPDF1B was located in the mitochondria. The plasmids pCAMBIA1300-35S: EuPDF1B-GFP and pCAMBIA1300-35S: COX-RFP were simultaneously transformed into 3-week-old A. thaliana mesophyll cell protoplasts, while the plasmids pCAMBIA1300-35S: GFP and pCAMBIA1300-35S: RFP were transformed into 3-week-old A. thaliana mesophyll protoplasts as controls. Then, the protoplasts of A. thaliana were cultured overnight at 23 °C, and the fluorescence distribution was observed and photographed by laser confocal scanning microscopy.

QRT‒PCR analysis

The accuracy of the RNA-seq results was verified by a qRT‒PCR system with RealStar Fast SYBR qPCR Mix (Genestar, Beijing, China). β-Actin and EuActin31 were used as internal reference genes for tobacco and E. ulmoides, respectively. The qRT‒PCR products were amplified as follows: 95 °C for 3 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. At least three independent biological replicates and three technical replicates were performed for each sample. The relative gene expression was calculated as described previously32.

GUS histochemical staining analysis

The transgenic tobacco tissues were immersed in GUS staining solution and incubated at 37 °C for 24 h. The tissues were decolorized with 70% ethanol to completely remove chlorophyll and observed and photographed with a stereomicroscope (Nikon, Japan).

Microscopic observation

The chloroplasts were analysed by transmission electron microscopy according to the methods described by Jung21. Each sample was washed with phosphate-buffered saline (pH 7.4) and fixed with 2.5% glutaraldehyde for 12 h, followed by 2% (w/v) osmium tetroxide for 2 h. After dehydration, the samples were sequentially embedded in EMbed 812 resin, polymerized into blocks after 3 days, and then sliced to 80 nm thickness by ultramicrosectioning. The chloroplasts were stained with 2% uranyl acetate and reynolds lead citrate for 10 min and 2 min, respectively, and then observed by transmission electron microscopy, after which the images were preserved.

Statistical analysis

Statistical analyses were performed using SPSS software, and the significance of differences between the control and transgenic plants was analyzed using Student’s t-test. A p value of 0.05 was considered to be significant. Statistical significance was tested using Duncan’s test at 0.05 probability levels.