Functional Identification of the Isopentenyl Diphosphate Isomerase Gene from Fritillaria unibracteata

Abstract

Isopentenyl diphosphate isomerase (IPI) is a key enzyme in the synthesis of isoprenoids. In this paper, the in vivo biological activity of the IPI gene from Fritillaria unibracteata (FuIPI) was investigated. Combining a color complementation experiment with High-Performance Liquid Chromatography analysis showed that the FuIPI gene could accumulate β-carotene in Escherichia coli, and Glu190 was identified as a key residue for its catalytic activity. Bioinformatics analysis together with subcellular localization indicated that the FuIPI protein was localized in chloroplasts. Compared with wild-type Arabidopsis thaliana, FuIPI transgenic plants had higher abscisic acid content and strengthening tolerance to drought and salt stress. Overall, these results indicated that the FuIPI gene had substantial biological activity in vivo, hopefully laying a foundation for its further research and application in liliaceous ornamental and medicinal plants.

1. Introduction

The family Liliaceae, encompassing a variety of ornamental and medicinal plants, has attracted great attention from botanical researchers. Specifically, Fritillaria unibracteata Hsiao et K.C. Hsia. (F. unibracteata) has been used as a primary medicinal resource for Fritillaria cirrhosa D. Don [1], a traditional Chinese medicine, for thousands of years [2]. F. unibracteata contains various active ingredients with antitussive effects, such as isosteroidal alkaloids, steroidal alkaloids, terpenoids, and steroidal saponins [3,4,5]. In addition, it exhibits superior pharmaceutic quality among Fritillaria cirrhosa D. Don due to its antitussive and asthma-relieving effects with minimal toxicity and side effects [6]. However, the wild resources of F. unibracteata have become rare due to not only aggressive harvesting but also the low accumulation of active ingredients [7]. Meanwhile, it is challenging to cultivate F. unibracteata in low-altitude regions, possibly due to its specific adaptability [8]. Therefore, the imbalance between high demand and insufficient supply has triggered research on the biosynthetic pathway of the active ingredients in F. unibracteata, laying a foundation for the metabolic engineering of its active ingredients.

The biosynthetic pathways of steroidal alkaloids and terpenoids are primarily involved in the mevalonate-independent (MVA) pathway and the methyl-erythritol 4-phosphate (MEP) pathway [9]. Several biosynthetic enzymes, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and lycopene β-cyclase [10,11], have been identified as key catalysts in the MVA and MEP pathways. The checkpoint of both pathways is the isomerization reaction of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), catalyzed by the enzyme isopentenyl diphosphate delta isomerase (IPI). Heterologous overexpression of exogenous IPIs in Escherichia coli (E. coli) increases the production of carotene [12], lycopene [13], and astaxanthin [14], highlighting their positive roles in the accumulation of downstream products. Intriguingly, IPI activity demonstrated a linear relationship with isoprene emission factors in the leaves of oak species [15], indicating its possible involvement in isoprene biosynthesis by oak leaves. Moreover, overexpression of the IPI genes in Eucommia ulmoides improves trans-polyisoprene production [16]. Overall, it is reasonably predicted that IPI may be a key enzyme linking the MVA and MEP pathways.

In our previous work, we obtained the IPI gene from F. unibracteata (FuIPI) encoding a peptide of 274 residues [17]. An in vitro enzymatic assay revealed that the recombinant FuIPI protein could convert IPP to DMAPP [17]. Therefore, further confirmation of its activity in vivo is crucial to understand its biological function. In this study, E. coli was firstly used as a prokaryotic host to verify the in vivo activity of FuIPI through a color complementation experiment combined with High-Performance Liquid Chromatography (HPLC) analysis. Subsequently, the subcellular localization of FuIPI protein was determined to predict the metabolite trafficking pathway in plants. Then, the FuIPI transgenic Arabidopsis thaliana (A. thaliana) was constructed. The phenotypes and metabolite change in wild-type and transgenic plants were evaluated. As a result, its activity in a eukaryotic host lays a theoretical foundation for regulating the biosynthesis of active compounds in F. unibracteata at the molecular level. To our knowledge, this is the first report of the in vivo functional identification of genes encoding enzymes related to the MVA and MEP pathways from the family Liliaceae and it might provide information for biological research on IPI genes from other liliaceous plants.

2. Materials and Methods

2.1. Materials

2.1.1. Strain and Plasmid

E. coli containing recombinant plasmid pET28a-FuIPI, plasmid pTrc with pAC-BETA, and plasmid pBI121 with green fluorescent protein (GFP) label were stored in the laboratory. Agrobacterium tumefaciens (A. tumefaciens) GV3101 was purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and stored at −80 °C in the laboratory. The primers used in the study were designed with Primer Premier 5.0 software and biosynthesized by TSINGKE Co., Ltd. (Beijing, China).

2.1.2. Reagents and Plant Materials

Plasmid Mini Kit I D6943 (OMEGA Bio-tek, Guangzhou, China), 1-5^TM 2× High Fidelity Master Mix (TSINGKE Co., Ltd., Beijing, China), Mut Express II Fast Mutagenesis Kit V2 (Vazyme Biotech Company, Ltd., Nanjing, China), Gel Extraction Kit D2500 (OMEGA Bio-tek, Guangzhou, China), BCA Protein Assay Kit (Sangon Biotech Co., Ltd., Shanghai, China), Prime STAR HS DNA Polymerase (Takara Biotechnology Co., Ltd., Dalian, China), and Plant hormone abscisic acid ELISA kit (Enzyme-linked Biotech Co., Ltd., Shanghai, China) were used. E. coli TOP10 strain, wild-type A. thaliana (ecotype Columbia-0), and Nicotiana benthamiana (N. benthamiana, ecotype LAB) seeds were kept in the laboratory.

2.1.3. Experimental Apparatus

Veriti^TM96 Well Fast Thermal Cycler (Thermo Fisher, Waltham, MA, USA), HPLC (Agilent, Santa Clara, CA, USA), Laser confocal microscopy A1R+ (Nikon 1774059S, Tokyo, Japan).

2.2. Experimental Methods

2.2.1. Construction of pTrc-FuIPI Expression Plasmid and Expression Host

Using plasmid pET28a-FuIPI as a template, the FuIPI gene fragment was amplified by polymerase chain reaction (PCR) using a pair of primers, 5′-GCGGCCGCGAAAACCTGTATTTTCAG-3′ (NotI cleavage site in italics, this forward primer is designed based on the 3–21 bp upstream sequence from the initiation codon ATG) and 5′-CGGGATCCCGTTAAATCAGTTTATGAATGG-3′ (BamHI cleavage site in italics). Subsequently, the fragment was digested with NotI and BamHI, gel-purified, and then cloned into plasmid pTrc to construct the recombinant plasmid pTrc-FuIPI. In addition, the recombinant plasmid pTrc-FuIPI, pTrc, and pAC-BETA were used to transform E. coli TOP10 strain to generate various recombinant strains, respectively.

2.2.2. Color Complementation Experiment

After verification by sequencing, four types of E. coli TOP10 strains, containing plasmid pTrc-FuIPI, plasmids pTrc and pAC-BETA, plasmid pAC-BETA, and plasmids pTrc-FuIPI and pAC-BETA, were cultured on Luria–Bertani (LB) solid medium at 37 °C for 48 h, respectively. The single colonies were then picked up and cultured on LB solid medium containing 50 μg/mL ampicillin (Ap) and 50 μg/mL chloramphenicol (Chl) at 37 °C for 48 h. Finally, the color depth of the colonies was observed and compared.

2.2.3. Determination of β-Carotene Content

Four types of bacterial solutions were transferred from the forementioned section to 50 mL of liquid LB medium and incubated at 37 °C overnight. Subsequently, 2–3 mL were taken and incubated at 28 °C and 200 rpm for 48 h to allow the β-carotene to fully accumulate. The β-carotene concentrations in E. coli TOP10 colonies were measured by HPLC analysis, as described previously [11]. The E. coli TOP10 cells were homogenized in methyl alcohol with 10% tetrahydrofuran and 0.01% dibutylhydroxytoluene, centrifuged, and filtered using a 0.45 μm microporous membrane [11]. For HPLC detection, the reversed-phase column Hypersil ODS2 (4.6 mm × 200 mm, 5 μm) was used with a mobile phase of methanol containing 10% tetrahydrofuran (volume fraction) and 0.0025% butylated hydroxytoluene. The injection volume was 10 μL with a flow rate of 1 mL/min. The column temperature was set at 30 °C and the detection wavelength was set at 448 nm.

2.2.4. Site Mutation at Glu190 Residue

As described by Song et al. [18], the codon GAA (Glu190) residue was replaced by GCG (Ala) residue using a pair of primers (5′-GAACATGCGCTGGATTATCTGCTGTTTATTGTTCG-3′ and 5′-GATAATCCAGCGCATGTTCACCCCATTTACC-3′). The site mutation was performed according to the site mutation kit using the plasmid pTrc-FuIPI as a template. The color complementation experiment of mutated pTrc-FuIPI E190A was then conducted using the same methods as for pTrc-FuIPI.

2.2.5. Subcellular Localization of FuIPI Protein

Using plasmid pET28a-FuIPI as a template, the FuIPI fragment was amplified by PCR with primers 5′-GAATTCATGGCAGCCGGGAGCGTT-3′ (EcoRI cleavage site in italics) and 5′-CGCGGATCCGCGAATCAGTTTATGAATGG-3′ (BamHI cleavage site in italics). In order to construct the fusion expression plasmid containing FuIPI and GFP fluorescent label, the stop codon (TAA) of the FuIPI gene was not included in the PCR product. Following the method described in Section 2.2.1, FuIPI was cloned into plasmid pBI121 (with GFP fluorescent label) and transformed into A. tumefaciens, which was then uniformly cultured on LB solid medium (containing 50 μg/mL gentamicin and 50 μg/mL kanamycin) at 30 °C for 2–3 days. Then, ten single colonies were randomly picked up and cultured for 24 h by shaking. After colony PCR verification, recombinant A. tumefaciens with plasmid pBI121-FuIPI was obtained.

A total of 2 mL of recombinant bacteria GV3101 containing pBI121-FuIPI was transferred and injected into the backs of N. benthamiana leaves and cultured for 2 days in low light. Fluorescence was observed using confocal laser microscopy. The excitation and emission wavelengths for chloroplast autofluorescence were set to 640 nm and 675 nm, respectively. The excitation and emission wavelengths for GFP were 488 nm and 510 nm, respectively.

2.2.6. Construction and Identification of FuIPI Transgenic A. thaliana

To analyze the functional roles of the FuIPI gene in transgenic plants, the FuIPI gene was overexpressed in wild-type A. thaliana. The FuIPI gene was first amplified by PCR using primers 5′-GCGGGTCGACGGTACCATGGCAGCCGGGAGCGTT-3′ and 5′-TAGACATATGGGTACCTTAAATCAGTTTATGAATGGTTTTC-3′ (KpnI cleavage site in italics), and subsequently ligated to obtain recombinant plasmid pCambia2301-FuIPI under the control of 35S promoter. The recombinant plasmid was transformed into A. tumefaciens and introduced into A. thaliana using the flower dip method [19]. The transgenic seeds were harvested and screened on 1/2 MS medium containing 50 μg /mL kanamycin. The lines showing positive growth were selected and cultivated in nutrient soil for two weeks. Leaves were used for the extraction of total DNA and RNA. DNA-based molecular verification of FuIPI transgenic plants was performed using primers 5′-CCTATTCTGCGTCTGCGTTCTT-3′ and 5′-TATCTGCAACTTCCTGCAGGG-3′. Meanwhile, the expression of the FuIPI gene in transgenic plants was analyzed by real-time PCR using primers 5′-AGCCATCCGCTGTATCGT-3′ and 5′-CATCGCTCGGTGCTTTAT-3′. The reference gene for real-time PCR was the Arabidopsis 18S rRNA gene (NC_003074.8) and the primers were 5′-CAGTCGGGGGCATTCGTATTT-3′ and 5′-CAGCCTTGCGACCATACTCC-3′.

2.2.7. Phenotypic Observation of FuIPI Transgenic Plants under Stress

Fourteen-day-old wild-type and FuIPI transgenic A. thaliana seedlings were subjected to natural drought and salt stress (120 mM NaCl solution), while the control group was watered with the same amount of water at the same frequency. After 21 days, the phenotypes of the plants were observed and analyzed, including plant height, fresh weight, leaf length, leaf width, etc. The materials were collected, frozen with liquid nitrogen, and stored at −80 °C.

2.2.8. Determination of Abscisic Acid (ABA) Content

Under no stress, drought stress, and salinity stress, 0.5 g of wild-type and FuIPI transgenic plants were weighed, ground with liquid nitrogen, extracted with 80% methanol at 4 °C for 16 h, and centrifuged at 10,000 rpm and 4 °C for 20 min, respectively. The extraction process was repeated twice, with the second extraction lasting 2 h. The collected supernatants were used to remove methanol by spin evaporation at 42 °C and subsequently extracted three times with ethyl acetate. The ABA fraction was dissolved in 0.8 mL methanol and assayed by an ELISA kit, according to the standard procedure.

3. Result

3.1. Functional Identification of FuIPI Gene in E. coli

Prior to the functional identification, sequence alignment between FuIPI protein and other IPIs with identified function was performed. As a result, based on the result of amino acid comparison and the classification of conserved structural motifs, the FuIPI protein was suggested to be included in the type I family. For instance, FuIPI displayed specific hits with two major motifs that are crucial for catalytic activities of the type I family. The first motif consisted of nine amino acids, ranging from Thr124 to Leu132. The other consensus motif with -WGEHE- and -DY- at the start and end points in the FuIPI protein was also detected from Trp186 to Tyr193. In addition, similar to other plant IPIs, FuIPI has a N-terminal extensions of 56 amino acids comparable with E. coli IDI (Figure 1).

The color complementation experiment was used for the functional identification of MEP pathway genes in various plants, including wheat [20] and Amomum villosum [21]. In this experiment, E. coli TOP10 cells along with cells containing a single plasmid of pTrc-FuIPI or pAC-BETA were used as negative controls. Two plasmids pTrc and pAC-BETA were co-transferred into E. coli TOP10 as positive controls. As a result, the regions 1, 4, and 5 (Figure 2), representing E. coli TOP10 cells, cells harboring plasmid pTrc-FuIPI, and cells harboring pAC-BETA, respectively, were unable to grow on LB medium with Ap and Chl due to the lack of the Ap resistance. Notably, the E. coli TOP10 cells containing plasmids pTrc-FuIPI and pAC-BETA in region 6 accumulated more yellow β-carotene than the positive controls (region 2) (Figure 2A). Overall, this visual method demonstrated that the FuIPI gene substantially accelerated β-carotene biosynthesis, confirming that FuIPI gene encoded the expected functional protein. Furthermore, the content of β-carotene in the E. coli TOP10 cells was determined by using HPLC. Neither E. coli TOP10 cells nor cells containing pTrc-FuIPI accumulated β-carotene, as they did not carry the plasmid pAC-BETA. A relatively small amount of β-carotene was observed in the E. coli TOP10 cells containing pAC-BETA (5.70 ± 0.094 μg/mL) and pTrc + pAC-BETA (16.56 ± 0.019 μg/mL), respectively. In contrast, the E. coli TOP10 cells co-transforming pTrc-FulPI and pAC-BETA revealed the highest β-carotene content (29.30 ± 0.4 μg/mL). Together with the visualization and HPLC analyses, it was further confirmed that the FuIPI gene might be limiting for carotenoid biosynthesis, as also reported by Cunningham and Gantt [22].

According to the sequence alignment and structural analysis, the Glu residue at position 190 was suggested to be a key residue in the active site of the IPI octahedron (Figure 1 in [17]). To test this, this reside was mutated to Ala residue. In the color complementation experiment (Figure 2A), E. coli TOP10 cells containing plasmids pTrc-FuIPI and pAC-BETA revealed the brightest color, followed by those containing plasmids pTrc-FuIPI E190A and pAC-BETA and, lastly, those with plasmids pTrc and pAC-BETA. This indicates that the E190A mutation caused a decrease in the accumulation of β-carotene. In addition, the β-carotene contents were detected using HPLC analysis (Figure 2B), the results of which indicate that the mutation caused a 15.30% reduction compared to cells containing plasmids pTrc-FuIPI and pAC-BETA. Overall, Glu190 residue was a key residue involved in the isomerization of FuIPI.

3.2. Subcellular Localization of FuIPI

iPSORT showed a putative mitochondrion-targeting peptide in the N-terminus of FuIPI, whereas ChloroP and Plant-mPLoc predicted that FuIPI is localized in chloroplasts with probabilities of 50% and 100%, respectively (Table 1). IPIs have been found to be localized in distinct compartments and vary among different plant species [23]. Considering the reaction catalyzed by FuIPI links MEP and MVA pathways and the subcellular localization of the MEP pathway in plants, it is a better prediction that FuIPI is localized in chloroplasts [24].

To verify this prediction, we constructed the recombinant plasmid PBI121-FuIPI labeled with GFP. Under laser confocal scanning, chloroplasts and GFP showed red and green fluorescence, respectively. As shown in Figure 3A, in the GFP control group, green fluorescence was uniformly distributed in the leaf cells of N. benthamiana without specificity. The subcellular localization of FuIPI protein was able to be revealed by the combined yellow fluorescence, indicating that FuIPI protein was localized in chloroplasts (Figure 3B) where the MEP pathway is localized.

3.3. Production of FuIPI Transgenic Plants

To verify the role of the FuIPI gene in influencing stress tolerance, recombinant plasmids containing FuIPI ORF, controlled by the CaMV 35S promoter, were constructed and transformed into A. thaliana. To validate the FuIPI transgenic plants, reverse PCR was performed using DNA as the template at first. As a result, the FuIPI fragment was detected in the FuIPI transgenic plants, while the wild-type plants exhibited negative PCR results (Figure 4A). In addition, the expression levels of the FuIPI gene were determined by real-time PCR using cDNA as the template. The FuIPI gene was substantially expressed in FuIPI transgenic plants, while it was not detected in wild-type plants (Figure 4B).

3.4. FuIPI Transgenic A. thaliana Strengthened Tolerance to Drought and Salinity Stress

The phenotypes of wild-type and FuIPI transgenic plants under various conditions were recorded to evaluate the function of the FuIPI gene in stress tolerance (Figure 5A). Although no obvious morphological difference was observed between the wild-type and transgenic plants, the FuIPI transgenic plants exhibited a higher fresh weight (163.18%), number of base leaves (126.41%), and leaf length (151.95%) than those in wild-type plants under normal condition, respectively (Figure 5B–E). These results indicate that the FuIPI gene positively affects plant growth. Moreover, it was observed that drought caused damage to the wild-type plants, including leaf yellowing and reduced growth, while the FuIPI transgenic plants exhibited slight enhancement in drought tolerance due to their higher fresh weight, number of base leaves (p < 0.05), leaf length, and leaf width compared to wild-type plants (Figure 5B–E). Intriguingly, FuIPI transgenic plants revealed significant tolerance to salinity stress, as their leaves remained green and growth remained vigorous. Compared with those of wild-type plants, the leaf length, leaf width, and fresh weight of FuIPI transgenic plants were significantly increased by 1.88- (p < 0.0001), 2.06- (p < 0.01), and 1.65-fold (p < 0.001), respectively. This higher number of leaves and increased leaf area contributed to more efficient photosynthesis and enhanced plant metabolism. In summary, the FuIPI gene can enhance tolerance to drought and salinity stress.

3.5. Overexpression of the FuIPI Gene Enhanced the Accumulation of ABA Content

The standard curve for ABA was determined with the equation y = 0.0191x + 0.1018 (R² = 0.995). Using this standard curve, the ABA content was measured. As shown in Figure 6, the ABA content in FuIPI transgenic plants was slightly higher than that in wild-type plants. Under drought conditions, the ABA content in FuIPI transgenic plants was 2.50 times that in wild-type plants (p < 0.0001), while the ABA concentration in FuIPI transgenic plants was also 1.50 times (p < 0.05) that in wild-type plants under salinity stress. Previous studies have confirmed that ABA is a stress-responsive plant hormone involved not only in tolerance to environmental stress [11] but also in plant development [25]. Therefore, these results indicated that the overexpression of the FuIPI gene could promote the accumulation of ABA, thereby improving the drought and salt tolerance of plants.

4. Discussion

In plants, the biosynthesis of isoprenoid-type metabolites occurs in three main stages [9]. In the upstream stage, the basic five-carbon unit IPP is derived from the MVA pathway in cytoplasm or MEP pathway in plastids. IPP is then isomerized to DMAPP by the catalysis of IPI. In the middle stage, IPP and DMAPP are sequentially condensed to yield intermediates such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP). Finally, in the downstream stage, these intermediates are converted into various isoprenoid-type metabolites, including steroidal alkaloid, artemisinin, chlorophyll, β-carotene, and ABA. Therefore, IPP and DMAPP serve as essential materials that connect the upstream MVA/MEP pathway with the downstream steroid biosynthesis pathway. IPI is proposed to catalyze a checkpoint reaction, providing the basic materials necessary for isoprenoid biosynthesis. We identified the in vivo function of the FuIPI gene through heterologous expression in prokaryotic and eukaryotic systems.

In line with previous studies, IPIs consist of two types (type I and II). To date, only type I IPIs have been found in plants, implying that the divergent functions of two types might occur before the differentiation of plants. We used two methods to confirm that FuIPI belonged to type I. At first, we compared the sequence identities of FuIPI with IPIs from other species. The top three species showing the highest similarity with FuIPI were Zingiber officinale, Vigna unguiculata, and Glycine max. The high similarity between FuIPI and other type-I IPIs from higher plants suggests that it is attributed to type I. Second, type-I IPIs have a conserved C residue in the TNTCCSHPL motif and a conserved E residue in the WGEHEXDY motif. These highly conserved residues are critical for the catalytic activity of the enzyme. FuIPI also contained the highly conserved C (124–132 aa) and E (186–193 aa) residues in the two motifs, strongly suggesting that it belongs to the type-I IPI family (Figure 1). As shown in Figure 2, in the color complementation experiment, the strains containing both pTrc-FuIPI and pAC-BETA appeared dark yellow on the medium, while those containing pTrc and pAC-BETA were light yellow, indicating that the expression of the FuIPI gene led to greater accumulation of β-carotene. HPLC analysis indicated that the FuIPI gene in E. coli enhanced β-carotent production by 5.14-fold higher than that in the control group.

Previous studies on IPI from E. coli have indicated that the active center of IPI bound to metal ions through a distorted octahedral coordination geometry composed of His25, His32, His69, Glu114, and Glu116 residues [26]. Among these, Glu116 (numbering based on E. coli IPI) was directly involved in the protonation of IPP to DMAPP. Therefore, the homologous Glu190 residue in FuIPI was hypothesized to be a key residue and was, thus, mutated to Ala. According to the color complementation test, the strain containing pTrc-FuIPI E190A was darker than those containing plasmids pTrc and pAC-BETA but lighter than those containing plasmids pTrc-FuIPI and pAC-BETA. The mutant resulted in a 15.30% decrease in β-carotene content compared with the E. coli strain containing plasmids pTrc-FuIPI and pAC-BETA. These results were consistent with our hypothesis that Glu190 in FuIPI was one of the key amino acids involved in the catalytic activity.

Many plants contained two type-I IPI isozymes with diversified subcellular localizations. Specifically, both IPIs from rice are localized in endoplasmic reticulum, peroxisomes, and mitochondria, whereas OsIPPI2 was also found in plastids [27]. A. thaliana has two IPIs that may be present in mitochondria and plastids, respectively [28], while IPIs from Nicotiana tabacum might function in cytosol and plastids [29], respectively. Intriguingly, most known plant type-I IPPs appeared to have an N-terminal extension that was associated with organellar localization [27,28,29]. Sequence analysis in Figure 1 indicated that the N-terminal extension of FuIPI shared an identity of more than 40% with that of OsIPPI2 (a plastid-localized IPI) while sharing only an identity of less than 20% with that of OsIPPI1 (a nonplastid-localized IPI) [27]. The subsequent bioinformatic prediction and subcellular localization of FuIPI confirmed its localization within the plastids, resembling that of IPIs from grape, Nicotiana tabacum, and rice [27,29,30]. The flow-controlled function of plastidial IPI for different downstream isoprenoid products, especially for those in plastids, has been highlighted by Pankratov et al. [31]. Hopefully, FuIPI could, thus, be a candidate gene for the metabolic engineering and synthetic biology of isoprenoids in plastids.

ABA has been implicated in plant stress tolerance, growth and development, and hormone responses [11,25]. By specifically binding to the cis-regulatory elements, such as DREBs and AREBs, in the promoters of genes involved in the ABA-related signaling pathway, ABA triggers various physiological processes and metabolic changes that enable whole tissues to acquire drought and salinity tolerance [32,33]. In plants, ABA is biosynthesized in plastids mainly from the β-carotene precursor, so an increase in E. coli overexpressing the FuIPI gene might enhance ABA biosynthesis. It was observed that the FuIPI transgenic plants contained an ABA content 2.50- and 1.50-fold higher than that in wild-type plants under drought and salinity stress, respectively. Considering the importance of ABA in the stress response of plants, it was particularly interesting to explore the phenotype of transgenic plants under these conditions. As a result, FuIPI transgenic plants exhibited slight enhancement in drought tolerance and significant enhancement in salinity tolerance compared with wild-type plants. Previous studies by our lab [11], along with findings from Marusig and Tombesi [34], have revealed that ABA correlates with improved drought and salinity tolerance in plants. Combined with current results and previous reports, the enhanced drought and salinity tolerance of transgenic plants could be attributed to its increased ABA content. Intriguingly, the FuIPI transgenic plants revealed higher biomass, including a greater fresh weight, higher number of base leaves, and longer leaf length than that of wild-type plants under no stress. This might imply that FuIPI would play a role in balancing plant growth and stress tolerance. A similar balance between plant growth and drought tolerance was also observed in plants overexpressing the Cassia tora DXR1 gene, possibly due to the regulation of ABA and chlorophyll biosynthesis [11]. Furthermore, the IPI genes from Aconitum carmichaelii, Dipsacus asperoides, and Artemisia annua have been proposed to play roles in the biosynthesis of diterpenoid alkaloids [35], triterpenoid saponin [36], and artemisinin [37], respectively. Therefore, in vivo functional identification of the FuIPI gene supported its role in the biosynthesis of isoprenoids, including β-carotene and ABA. It is hypothesized that FuIPI might be applied to accumulate isoprenoid-derived compounds in medicinal plants, to improve the nutritional qualities of crops by enhancing the accumulation of compounds such as β-carotene, and to improve the yield of crops by increasing adaptability to environmental stress as well.

Although the phenotypes of FuIPI transgenic plants can be largely linked to altered ABA levels, future research remains necessary. First, in addition to the aerial tissues, the underground tissues should also be evaluated for the drought and salinity tolerance of FuIPI transgenic plants, given that ABA exerts a wide influence on various plant tissues. Second, a detailed investigation into the expression of downstream genes that are involved in the ABA-dependent signaling pathway is required to comprehensively understand the role of the FuIPI gene in drought and salinity tolerance. Moreover, in order to explore the potential application of the FuIPI gene in various fields, different chassis, such as crops and medicinal plants, will be used in upcoming research.

5. Conclusions

This study demonstrates that FuIPI is involved in the biosynthesis of isoprenoids by heterologous expression in both prokaryotic and eukaryotic cells. The site mutation confirmed that Glu190 was one key residue related to the catalytic reaction. The subcellular localization of FuIPI in chloroplasts provided a challenge to monitor the crosstalk between MVA and MEP pathways, which were associated with diverse biological processes. The tolerance to drought and salinity stress could be augmented by the overexpression of the FuIPI gene, as indicated by the improving accumulation of ABA content. Overall, our studies extend the IPI members of the family Liliaceae, which is rich with ornamental and medicinal plants. In addition, our current work will contribute to understanding the molecular mechanisms of promoting isoprenoid-related accumulation and enhancing stress tolerance via the overexpression of the FuIPI gene.

Author Contributions

X.Y., J.C. (Jiao Chen), H.Y., X.H., J.C. (Jieru Chen), Z.M., J.Z. and H.L. performed the experiments. X.Y., J.C. (Jiao Chen), H.Y., X.H., J.C. (Jieru Chen) and Z.M. analyzed the data. H.L. and J.Z. contributed to the materials and analysis tool. H.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The full-length coding sequence (CDS) of FuIPI was submitted to GenBank databases with the accession number PQ058665. In this study, the codon-optimized FuIPI, the original gel of Figure 4A, and other original data are included in the Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Table

Figure 1. The amino acid sequence alignment of FuIPI with IPIs in other species. The amino acid accession numbers are as follows: Arabidopsis thaliana IDI1 At5g16440, Arabidopsis thaliana IDI2 At3g02780, Salvia miltiorrhiza EF635967, Eucommia ulmoides AB041629, Nicotiana tabacum BAB40973.1, Oryza sativa IPI1 AK065871, Oryza sativa IPI2 NM_001062082, and E. coli NP_417365. The conserved TNTCCSHPL and WGEHEXDY motifs were indicated by black lines, while the key Glu190 was indicated by regular triangles. The N-terminal extension was indicated by dashed line.

Figure 2. Color complementation experiment and HPLC detection of six E. coli TOP10 colonies. (A) Color complementation experiment; (B) β-carotene contents detected by HPLC. One-way ANOVA was used for analysis. All the experiments had at least three biological replicates. Regions 1, 2, 3, 4, 5, and 6 represent E. coli TOP10, E. coli TOP10 containing plasmids pTrc and pAC-BETA, E. coli TOP10 containing plasmids pTrc-FuIPI E190A and pAC-BETA, E. coli TOP10 containing plasmid pTrc-FuIPI, E. coli TOP10 containing plasmid pAC-BETA, and E. coli TOP 10 containing plasmids pTrc-FuIPI and pAC-BETA, respectively. Different lowercase letters represent significant differences between pairs (p [less than] 0.05).

Figure 3. The subcellular localization of FuIPI in N. benthamiana leaves: (A) represents 35S: GFP, while (B) represents 35S: FuIPI-GFP.

Figure 4. Identification of FuIPI transgenic plants: (A) represents the PCR result using DNA as the template and (B) represents the PCR result using cDNA as the template.

Figure 5. Phenotypic analysis of A. thaliana under various conditions. (A) Phenotypes of plants under various conditions. WT and FuIPI represent wild-type and FuIPI transgenic plants, respectively. The plants from left to right in Figure 5A represent those under no stress, drought stress, and salinity stress, respectively. (B–E) Represent various characteristics of plants, including fresh weight, base leaf number, leaf length, and leaf width, respectively. One-way ANOVA was used to perform statistical analysis. All the experiments had at least three biological replicates. *, **, *** and **** represented statistical significance of p [less than] 0.05, p [less than] 0.01, p [less than] 0.001, and p [less than] 0.0001, respectively.

Figure 6. ABA content in plants under no stress, drought stress, and salinity stress. WT and FuIPI represent wild-type and FuIPI transgenic plants, respectively. One-way ANOVA was used for statistical analysis. All the experiments had at least three biological replicates. * and **** represented statistical significance of p [less than] 0.05 and p [less than] 0.0001, respectively.

Table 1

Prediction of FuIPI subcellular localization.

Software	Prediction Result
iPSORT	mitochondrial targeting or chloroplast transport peptide
Plant-mPLoc	chloroplast

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10080887/s1, Supplementary Material S1: codon-optimized FuIPI; Supplementary Material S2: The original gel of Figure 4; Supplementary Material S3: The original data of Figure 2B; Supplementary Material S4: The original data of Figure 4B; Supplementary Material S5: The original data of Figure 5B; Supplementary Material S6: The original data of Figure 6.

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