Expression and promoter analysis of MEP pathway enzyme-encoding genes in Pinus massoniana Lamb

Plant materials and elicitor treatments

Seeds of P. massoniana were collected from the National P. massoniana Superior Variety Base of Xinyi, in Guangdong, China. Two-year-old P. massoniana plants were transplanted into pots containing a soil mixture (peat:perlite:vermiculite, 3:1:1 (v/v)) at 24 °C under a 16-h light/8-h dark photoperiod. Plants at similar growth stages were selected for different tissue collections and subsequent treatments.

Four elicitor treatments were applied (10 mM H₂O₂, 100 µM methyl jasmonate (MeJA), 500 μM ethephon (ETH), and 1 mM salicylic acid (SA)). It is important to note that MeJA does not dissolve easily in water; however, it is soluble in ethanol. Thus, MeJA was dissolved in 5 mL ethanol and then diluted with distilled water to volume in a 1 L volumetric flask. Finally, these solutions were sprayed onto the needles of the seedlings, which were exposed to the treatments for 0 h, 3 h, 12 h, and 24 h. Subsequently, the needles were collected for RNA extraction, where the samples treated with only the corresponding solvent were used as controls. Each treatment was repeated three times, which served as biological replicates. All collected samples were frozen in liquid nitrogen and stored at −80 °C for DNA and RNA extraction.

Total RNA, gDNA extraction, and cDNA synthesis

The total RNA was extracted by the RNAprep Pure Kit (DP441; Tiangen Biotech, Beijing, China). A NanoDrop 2000 instrument (Thermo Fisher, Waltham, MA, USA) and 1.2% agarose gel electrophoresis were employed to detect the concentration, integrity and purity of the total RNA.

The cDNA (20 μL) was synthesized using a first-strand cDNA synthesis kit (11141; Yeasen Biotech, Shanghai, China) with 1,000 ng total RNA.

The genomic DNA (gDNA) was extracted using a Plant Genomic DNA Kit (DP320; Tiangen Biotech, Beijing, China). The gDNA concentration and purity were measured using a NanoDrop 2000 instrument, and the gDNA integrity was estimated via 1.2% agarose gel electrophoresis.

Cloning of MEP pathway enzyme-encoding genes and sequence analysis

The ORF primers used for cloning the PCR are shown in Table S1. The PCR products were cloned into the pUC19 vector and Sanger sequencing was performed to verify the correction of the enzyme-encoding genes of the MEP pathway. The physicochemical properties of the MEP pathway enzyme proteins were analyzed using the ExPASy ProtParam online program (https://web.expasy.org/protparam/). Transmembrane domain analysis was performed using TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM). Subcellular localization was predicted using the PSORT (https://psort.hgc.jp/) and Cell-PLoc version 2.0 programs (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). Multiple sequence alignment was performed using the Blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and DNAMAN programs. Conserved domain analyses were performed using the InterPro online program (http://www.ebi.ac.uk/interpro/).

Subcellular localization

The vector construction and transient transformation were conducted as described previously (Zhu et al., 2021). Briefly, the CDS (Coding sequence) regions were inserted into a pBI121-GFP vector (35S::PmDXS1-GFP, etc.) that fused a green fluorescent protein (GFP). The primers used for vector construction are shown in Table S2. The fusion vectors were transformed into the Agrobacterium tumefaciens strain GV3101(pSoup-p19). Agrobacterium strains were grown in LB media for 36 h at 28 °C and then suspended in infiltration media (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgCl₂, and 150 μM acetosyringone) for transient transformation. The suspension cells were infiltrated into the young leaves of 30- to 40-day-old Nicotiana benthamiana. Then the infiltrated tobacco plants were maintained under a 16 h light/8 h dark photoperiod at 22 °C for 48 h. The GFP and chloroplast fluorescence signals of the tobacco leaves were obtained using an LSM710 confocal laser scanning microscope (Zeiss, Jena, Germany).

Isolation and sequence analysis of MEP pathway enzyme gene promoters

The promoter sequences were isolated using hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) from the genomic DNA of P. massoniana, as described by Liu & Chen (2007). The primers used for hiTAIL-PCR are shown in Table S3. Preamplification (20 μL), primary TAIL-PCR (50 μL), and secondary TAIL-PCR (50 μL) were performed as described by Liu & Chen (2007). The PCR products were cloned into the pUC19 vector and Sanger sequencing was performed. The structural analysis of the enzyme gene promoters in the MEP pathway were preformed using PLACE (https://www.dna.affrc.go.jp/PLACE/?action=newplace) and PlantCARE programs (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Expression analysis of MEP pathway enzyme-encoding genes

Three biological replicates (plant samples) and three technical replicates (qPCR reactions) were used in expression analysis. In detail, for different tissues and elicitor treatments, corresponding samples were collected from three independent plants as biological replicates. In the qPCR assay, each reaction well was replicated three times (they contained the same cDNA template and primers), as technical replicates. The qPCR was conducted using a StepOne Plus instrument (Applied Biosystems, Waltham, MA, USA). The SYBR Green Mix (11184; Yeasen Biotech, Shanghai, China) and an internal control gene TUA (alpha-tubulin) were employed in the qPCR reactions (Zhu et al., 2019). The specific primers are shown in Table S4. Each PCR mixture (10 μL) consisted of 1 μL cDNA (20× dilution), 5 μL of SYBR Green Mix, 0.4 μL of each primer (10 μM), and 3.2 μL of ddH2O. The amplification was conducted as described previously (Zhu et al., 2021). The relative expression was computed by the 2^−ΔΔCt method (Livak & Schmittgen, 2001).

Functional complementation

The EcAB4-2 strain of Escherichia coli was a dxs mutant, which was also MVA auxotrophy (Perez-Gil et al., 2012). This indicated that the EcAB4-2 strain survived only in the medium with exogenous MVA. The EcAB4-2 strain was used to test the functions of the PmDXS1, PmDXS2, and PmDXS3 genes. The ORFs of the three PmDXSs were inserted into a pET-28a prokaryotic expression vector, with the primers used for vector construction shown in Table S2. These constructed vectors were used to transform the EcAB4-2 strain. The transformants were selected on Luria-Bertani (LB) solid medium with 25 μg/mL kanamycin, 15 μg/mL chloramphenicol, and 1 mM mevalonate (MVA).

Cloning and sequence analyses of MEP pathway enzyme-encoding genes

The ORF (open reading frame) sequences of the MEP pathway enzyme genes were obtained using the online program ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) based on unassembled full-length transcripts from the Iso-seq data. PCR and Sanger sequencing verified the ORFs of eleven enzyme genes in the MEP pathway (PmDXS1, PmDXS2, PmDXS3, PmDXR, PmMCT, PmCMK, PmMDS, PmHDS, PmHDR1, PmHDR2, and PmIPPI) comprised of 2,154 bp, 2,217 bp, 2,223 bp, 1,440 bp, 954 bp, 1,218 bp, 726 bp, 2,232 bp, 1,464 bp, 1,458 bp, and 939 bp, respectively (Fig. 2). Eleven CDSs (Coding sequences) of MEP pathway enzyme-encoding genes from P. massoniana were submitted to the NCBI (accession number MW892440–MW892450) and are displayed in Table S1. The amino acid lengths, molecular weights, isoelectric points (pI), and transmembrane domains are shown in Table 1.

The results of multiple alignments in the NCBI database revealed that the amino acid sequences of the eleven MEP genes from P. massoniana possessed a high homology with other Pinaceae plants. The results of multiple alignments with other plants (except for Pinaceae) revealed that the identities ranged from 59.37% to 88.97%, in which PmDXS1, PmDXR, and PmHDS shared higher identities (ranging from 73.88% to 88.97%), and where PmCMK showed the lowest identity (ranging from 59.37% to 74.61%) (Fig. S1). Furthermore, the alignment results suggested that PmDXS1 and PmDXS2 belonged to type II DXS, and PmDXS1 belonged to type I DXS. Similarly, between the two HDR homologs, PmHDR1 and PmHDR2 belonged to type II and type I, respectively. The results of phylogenetic grouping clearly showed three DXS (Fig. 3A) and two HDR (Fig. 3B) homologous proteins clustered into independent clades.

Conserved domain analyses are shown as a cartoon in Fig. 4. The results indicated that PmDXS1, PmDXS2, and PmDXS3 proteins had highly similar domains, including a thiamin diphosphate-binding domain at the N-terminal, and a transketolase domain at the C-terminal. The PmDXR protein contained three reductoisomerase domains at 87-214aa, 228-311aa, and 343-463aa, in which the first reductoisomerase domain at the N-terminal was also considered a NAD(P)-binding domain. A nucleotide-diphospho-sugar transferase domain was found at the C-terminal of the PmMCT protein. The PmCMK protein contained two GHMP kinase domains at 175-229aa and 253-353aa. A MECDP synthase domain (also known as the IspF/YgbB domain), was found at the C-terminal of the PmMDS protein. The PmHDS protein showed a dihydropteroate (DHP) synthase domain at the N-terminal and a nitrite and sulphite reductase 4Fe-4S domain at the C-terminal. Both PmHDR1 and PmHDR2 proteins revealed a LytB/IspH domain. The PmIPPI protein contained a NUDIX hydrolase domain at 129-281aa. The conserved domains between the MEP pathway enzyme proteins were crucial for their specific biological functions.

Eleven MEP pathway enzymes in P. massoniana target the N. benthamiana chloroplast

PSORT and Cell-PLoc were used for the prediction of subcellular localization, where according to both methods, the eleven MEP pathway enzyme proteins had the same predicted localizations within the chloroplast (Table 2). Furthermore, a transient expression experiment was performed to further explore the subcellular localization characteristics of the eleven MEP pathway enzyme proteins. The GFP fluorescent signals of the eleven MEP pathway enzyme proteins were observed to overlap with the chloroplast red fluorescence signals. This indicated that the subcellular localizations of the eleven MEP pathway enzyme proteins were targeted to the chloroplast (Fig. 5).

Expression patterns of MEP pathway enzyme-encoding genes in different tissues

The qPCR was performed to investigate the tissue-specific expressions of the eleven MEP genes in young needles, old needles, stems, and roots of P. massoniana. Tissue-specific expression patterns suggested that most of the MEP genes had higher expression levels in the needles. PmDXS1, PmDXR, PmMCT, PmCMK, PmMDS, and PmIPPI showed the highest transcript levels in old needles, whereas PmHDS and PmHDR2 revealed the highest transcript levels in young needles (Fig. 6). Interestingly, three DXSs and two HDRs homologous genes had different expression patterns. PmDXS2 and PmDXS3 had significantly high transcript levels in the roots, which was quite different from PmDXS1. In particular, the expression of PmDXS2 in the roots were 10.45-fold, 5.84-fold, and 7.34-fold higher in young needles, old needles, and stems, respectively. PmHDR1 showed the highest expression levels in roots, while PmHDR2 showed quite low expression levels in stems and roots. The expression of PmHDR2 in young needles was 31.58- and 176.47-fold higher than that in stems and roots, respectively.

The results of the eleven MEP gene expression levels in the same tissue revealed that most had similar patterns in both young and old needles (Fig. 7). PmHDR2 showed the highest transcript level in the needles, followed by PmMDS and PmDXS2. PmDXS1 revealed the highest transcript level in the stems, followed by PmHDR1 and PmDXS2. Finally, PmHDR1 had the highest expression level in the roots, with PmMDS being the second highest, and PmHDR2 the lowest.

Expression patterns of MEP pathway enzyme-encoding genes under different elicitor treatments

The qPCR was performed to investigate the expression patterns of the eleven MEP genes under ETH, MeJA, SA, and H₂O₂ elicitor treatments. For the ethephon (ETH) treatment, except for PmMDS, the other ten MEP genes had significantly high expression levels at 3 h (1.62- to 4.54-fold). However, the expression of PmMDS was substantially downregulated at 12 h and 24 h, particularly at 24 h (0.56-fold) (Fig. 8).

For the H₂O₂ treatment, the PmCMK expression level showed no significant change across all time points. The expressions of PmDXS1, PmDXR, PmMCT, PmMDS, PmHDS, and PmHDRs were considerably downregulated following the H₂O₂ treatment. The expression levels of PmDXS2, PmDXS3, and PmIPPI were strongly upregulated at 3 h (11.48-, 1.64-, and 1.69-fold, respectively) (Fig. 9).

For the salicylic acid (SA) treatment (except for PmMCT and PmCMK), the expression levels of the other nine MEP genes had significantly high expression levels at 3 h (1.95- to 72.04-fold). Interestingly, the expression of PmCMK revealed a sharp downregulation following the SA treatment (>100-fold), while the expression of PmDXS2 showed a sharp upregulation at 3 h (72.04-fold) (Fig. 10).

For the methyl jasmonate treatment, the expression of PmMCT, PmCMK, and PmHDR1 were significantly downregulated at only 24 h (0.32-, 0.68-, and 0.32-fold, respectively). The expressions of PmDXR, PmMDS, PmHDS, PmHDR2, and PmIPPI were continuously downregulated, particularly at 24 h (0.20- to 0.49-fold). Interestingly, only PmDXS2 showed a significant upregulation (9.46-fold) (Fig. 11).

Cis-elements and putative binding sites of transcription factors found in MEP pathway enzyme-encoding gene promoters

Due to the lack of a P. massoniana reference genome, ten promoter sequences (except for PmHDS) were obtained using high-efficiency thermal asymmetric interlaced PCR. Sanger sequencing verified ten promoter sequences of the MEP pathway enzyme-encoding genes (PmDXS1, PmDXS2, PmDXS3, PmDXR, PmMCT, PmCMK, PmMDS, PmHDR1, PmHDR2, and PmIPPI), comprised of 1,714 bp, 735 bp, 853 bp, 1,212 bp, 577 bp, 1,654 bp, 1,431 bp, 1,276 bp, 820 bp, and 562 bp, respectively (Table S5).

The PlantCARE program was utilized to predict and analyze the cis-acting elements of these promoter sequences (Table S6). The ten promoters contained multiple typical cis-acting elements, including CAAT-box and TATA-box. Further, many light responsive cis-acting elements were predicted, such as ACE, ATC-motif, Box 4, G-box, GATA-motif, TCC-motif, TCT-motif, and GT1-motif. Three elements responded to the methyl jasmonate (MeJA) and salicylic acid (SA) elicitors, including the TCA-motif, CGTCA-motif, and TGACG-motif. Six phytohormone response-related cis-acting elements were identified, such as ABRE, AuxRR-core, TGA-element, GARE-motif, and TATC-box. The ARE and LTR elements were predicted to be related to anaerobic induction and low-temperature response, respectively.

The AP2/ERF, bHLH, bZIP, WRKY, and MYB target sites were identified via PLACE programs (Fig. 12 and Table S7). Numerous target sites of the five TF families were detected to be distributed in the ten promoters. The binding elements of MYB and WRKY TFs were the most widely distributed, while the bZIP binding sites were the least distributed. The EBOXBNNAPA and WRKY71OS elements, as bHLH and WRKY TF binding sites, respectively, appeared in all the promoters, and contained at least two binding sites. The PmCMK and PmDXS1 promoters contained twelve EBOXBNNAPA elements and twenty WRKY71OS elements, respectively. The MYBST1 and RAV1AAT elements, as MYB and AP2/ERF TF binding sites, respectively, appeared in nine promoters. These results suggested that AP2/ERF, bHLH, bZIP, WRKY, and MYB TFs might directly target the MEP pathway enzyme-encoding genes that affect terpenoid production in P. massoniana.

PmDXSs allowed the survival of the dxs mutant E. coli strain

In the functional complementation experiment, only the dxs mutant strains that expressed three PmDXS proteins from P. massoniana grew normally on the solid medium without exogenous MVA, respectively (Fig. 13). However, the dxs mutant strain and the mutant containing an empty pET-28a vector could not grow on the same medium (Fig. 13). This signified that the three PmDXS provided DXS enzyme activity for the strains.