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. 2026 Aug 7;178(4):e71059. doi: 10.1111/ppl.71059

The PmRDUF1‐PmMYB30‐PmLAS Regulatory Module Is Involved in Terpenoid Biosynthesis of Pinus massoniana

Luonan Shen 1,2, Sulin Wen 1, Lin Deng 1, Kui Zhou 2, Kun Yang 2, Guang Qiao 2, Yi Min 1, Lei Peng 1, Chaojin Wei 2, Zhengdong Cai 2, Xiaopeng Wen 2,✉
PMCID: PMC13448752  PMID: 42563601

ABSTRACT

The resin of masson pine ( Pinus massoniana L.) exhibits considerable economic value and primarily consists of diterpenoid resin acids. However, the upstream regulation of the key diterpene biosynthetic gene, PmLAS, remains largely unclear. In the present study, through yeast one‐hybrid screening, three PmMYB transcription factors were identified: PmMYB30, PmMYB68, and PmMYB74. Subsequent verification through dual‐luciferase assays substantiated that these transcription factors interact with the PmLAS promoter, thereby facilitating its transcriptional activation. Subcellular localization analysis indicated that all three PmMYBs are localized in the nucleus, and transcriptional activation assays in yeast further revealed that their C‐terminal regions are responsible for the transcriptional activation activity. Meanwhile, the transcript levels of three PmMYBs exhibited a significant positive correlation with resin yield in the xylem of 12‐year‐old masson pine trees. Moreover, the heterologous overexpression of these genes in tobacco resulted in a growth‐inhibiting phenotype, a marked increase in leaf glandular trichome numbers, and an elevated proportion of diterpenoids compared to wild‐type tobacco. To further elucidate the molecular regulation of the PmLAS gene by the transcription factor PmMYB30, yeast two‐hybrid library screening was performed to screen its interacting proteins. A RING‐type E3 ubiquitin ligase was identified from the screening results and designated PmRDUF1. Subsequent luciferase complementation imaging assays provided further evidence supporting the interaction between PmRDUF1 and PmMYB30. Furthermore, dual‐luciferase reporter assays demonstrated that this interaction suppresses the ability of PmMYB30 to activate the expression of PmLAS, forming a PmRDUF1‐PmMYB30‐PmLAS regulatory module. Collectively, our findings suggest that a complex transcriptional network governs diterpenoid biosynthesis in masson pine, offering new perspectives on the regulation of terpenoid metabolism.

Keywords: E3 ubiquitin ligase, Levopimaradiene synthase (PmLAS), MYB transcription factors, Pinus massoniana L., terpenoid biosynthesis

1. Introduction

Masson pine ( Pinus massoniana L.) is a prominent resin‐producing tree species indigenous to southern China (Liu et al. 2020; Bai 2022). Resin from masson pine is an economically important raw material, which serves as the feedstock for manufacturing chemical products, pharmaceuticals, coatings, and printing inks across multiple industrial sectors (Li and Chen 2024; Zhang et al. 2025). The masson pine resins' chemical composition consists primarily of monoterpenes, sesquiterpenes, and diterpenoids (Yatagai and Hong 1997; Fan et al. 2013; Wu et al. 2019). These compounds are synthesized via two distinct metabolic pathways: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway (Rohmer 2003).

The MVA pathway starts with acetoacetyl‐CoA thiolase (AACT) converting acetyl‐CoA to acetoacetyl‐CoA (Vollack and Thomas 1996; Dyer et al. 2009). Subsequently, a series of enzymatic reactions catalyzed by hydroxymethylglutaryl‐CoA synthase (HMGS; Nagegowda et al. 2004; Liao et al. 2020), hydroxymethylglutaryl‐CoA reductase (HMGR; Venkatachalam et al. 2009; Li et al. 2014), mevalonate kinase (MVK; Lluch et al. 2000), phosphomevalonate kinase (PMVK; Schulte 1999), and diphosphomevalonate decarboxylase (MVD; Zou et al. 2013) convert acetoacetyl‐CoA into the fundamental five‐carbon isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). HMGR is the rate‐limiting enzyme of the MVA pathway and represents a key regulatory point in plant terpenoid biosynthesis (Chappell et al. 1995; Liu et al. 2018).

The plastidial MEP pathway converts the substrates pyruvate and glyceraldehyde 3‐phosphate into IPP and DMAPP through the sequential action of 1‐deoxy‐d‐xylulose‐5‐phosphate synthase (DXS; Lichtenthaler 1999), 1‐deoxy‐d‐xylulose‐5‐phosphate reductoisomerase (DXR; Kuzuyama, Takagi, et al. 2000), 2‐C‐methyl‐d‐erythritol 4‐phosphate cytidylyltransferase (MCT; Kuzuyama, Takahashi, et al. 2000), 4‐diphosphocytidyl‐2‐C‐methyl‐d‐erythritol kinase (CMK; Rohdich et al. 2000), 2‐C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate synthase (MECPS; Kumar et al. 2012), 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase (HDS; Kim and Kim 2010), and 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate reductase (HDR; Adam et al. 2002). Within the MEP pathway, DXS and DXR are pivotal enzymes that play crucial roles in the biosynthesis of plant terpenoids (Walter et al. 2002; Zhang et al. 2018; Ye et al. 2023).

The biosynthesis of terpenoid backbone precursors is catalyzed by distinct synthases: Farnesyl pyrophosphate synthase (FPPS), geranyl pyrophosphate synthase (GPPS), and geranylgeranyl pyrophosphate synthase (GGPPS) catalyze the condensation of IPP and DMAPP to generate distinct isoprenoid precursors. Specifically, GPPS produces the C10 monoterpene precursor GPP, FPPS yields the C15 sesquiterpene precursor FPP, and GGPPS synthesizes the C20 diterpene precursor GGPP (Adiwilaga and Kush 1996; Bouvier et al. 2000; Burke and Croteau 2002). These precursors are then converted into a diverse array of final terpenoid products by terpene synthases (TPSs). The TPS‐d subfamily, which is specific to conifers, performs this critical catalytic step. In masson pine, (+)‐α‐pinene synthase, (−)‐α‐pinene synthase, and limonene synthase produce (+)‐α‐pinene, (−)‐α‐pinene, and limonene as their major products, respectively. The (−)‐β‐pinene synthase, however, is solely responsible for generating trace quantities of (−)‐β‐pinene (Wen et al. 2024). In addition, PmTPS4 primarily catalyzes the formation of α‐pinene, whereas PmTPS21 exhibits a bifunctional activity, producing both α‐pinene and longifolene (Liu et al. 2021).

Levopimaradiene synthase (LAS) is a key diterpene cyclase that plays a critical role in the biosynthesis of diterpenoid resin acids across diverse gymnosperm species. In Ginkgo biloba , a LAS gene had been cloned and functionally characterized, with the enzyme confirmed to catalyze the cyclization of GGPP into levopimaradiene (Schepmann et al. 2001). In Pinus taeda , PtTPS‐LAS utilizes GGPP as a substrate and produces a mixture of diterpene products, including levopimaradiene (45.5%), abietadiene (27.6%), neoabietadiene (22.4%), and palustradiene (4.5%; Ro and Bohlmann 2006). Our previous work showed that the expression of the levopimaradiene synthase (PmLAS) gene obviously affects the production of levopimaric acid, thereby affecting resin biosynthesis in masson pine (Shen et al. 2025). However, the underlying molecular regulatory mechanisms remain elusive.

To elucidate the molecular regulatory mechanisms of the PmLAS gene in masson pine, a yeast one‐hybrid (Y1H) screen was performed to identify transcription factors that regulate the PmLAS promoter. Candidate transcription factors were functionally characterized using pairwise Y1H validation, dual‐luciferase reporter assays, subcellular localization, and transcriptional activation assays. Subsequently, expression and correlation analyses of candidate genes were performed in masson pine with different resin yields. Moreover, candidate genes were heterologously overexpressed in tobacco, with changes in the transgenic lines analyzed using qRT‐PCR, metabolomics (UPLC‐MS/MS), scanning electron microscopy (SEM), and phenotypic measurements to assess their functional effects. Furthermore, the impact of protein–protein interactions on the regulatory pathway was investigated through yeast two‐hybrid (Y2H) library screening, luciferase complementation imaging assays, and additional dual‐luciferase experiments. This study aimed to provide new insights into the molecular regulatory network controlling PmLAS expression.

2. Materials and Methods

2.1. Y1H Assay

To construct the bait plasmid (pHIS2‐PmLAS), the promoter of the PmLAS gene was cloned into the pHIS2 vector. For yeast transformation, 480 μL of 50% PEG, 72 μL of 1 M LiAc, 10 μL of denatured salmon sperm DNA, and 5 μL of each plasmid (library plasmid and pHIS2‐PmLAS) were sequentially added to competent Y187 yeast cells. The mixture was incubated in a water bath at 30°C for 30 min and mixed by gentle inversion at intervals. Subsequently, 20 μL of DMSO was supplemented, followed by heat shock treatment at 42°C for 25 min. The cell suspension was centrifuged in a refrigerated microcentrifuge at 3582g for 30 s, then 1 mL of liquid YPDA medium was added, and the sample was incubated at 30°C for 1 h with shaking. After low‐speed centrifugation (64 g, 3 min), the supernatant was discarded. The pelleted yeast cells of each transformation reaction were resuspended in 200 μL of 0.9% NaCl solution and spread onto synthetic dropout solid medium SD/−Trp/−Leu/‐His supplemented with 60 mM 3‐amino‐1,2,4‐triazole (3‐AT). Plates were incubated at 30°C for 2 d in a constant‐temperature incubator. Single colonies were subjected to colony PCR, and amplicons from clones yielding a single product were sequenced by Sangon Biotech. The obtained sequences were aligned with the NCBI database to confirm their accuracy. For one‐to‐one interaction validation, candidate genes identified from the screening were individually cloned into the pGADT7 prey vector. Each of the resulting prey plasmids (pGADT7‐PmMYB30, pGADT7‐PmMYB68, and pGADT7‐PmMYB74) was then co‐transformed with the pHIS2‐PmLAS bait plasmid into Y187 yeasts. The co‐transformants were spotted onto SD selective medium without Trp, Leu, and His, containing 60 mM 3‐AT to assess interaction. A co‐transformation of pGADT7‐rec‐53 and pHIS2‐p53 served as the positive control. Table S1 contains a list of all primers used in this work.

2.2. Dual‐Luciferase Reporter Assay

The promoter sequence of the PmLAS gene was amplified using specific primers and cloned into the pGreen‐0800 vector via seamless cloning to generate the reporter construct pGreen‐0800‐proPmLAS‐Luc. The coding sequences of PmMYB30, PmMYB68, PmMYB74, and PmRDUF1 were individually cloned into the pGreen‐62SK vector. All constructs were transformed into Agrobacterium tumefaciens GV3101. Different plasmid combinations in bacterial suspensions were co‐infiltrated into tobacco (Nicotiana benthamiana) leaves. Following for luminescence imaging, a d‐luciferin potassium salt solution was applied to the leaves, and the fluorescence signal was captured using the PlantView100 (BLT). Luciferase activity was measured using a firefly luciferase reporter assay kit (Sangon Biotech) on a Varioskan LUX multifunctional microplate reader (Thermo Scientific).

2.3. Subcellular Localization

Specific primers (Table S1) were used to amplify the coding sequences of PmMYB30, PmMYB68, and PmMYB74. Each amplified fragment was inserted into the 35S‐eGFP vector via seamless cloning, generating the 35S‐PmMYB30‐eGFP, 35S‐PmMYB68‐eGFP, and 35S‐PmMYB74‐eGFP fusion constructs. Subsequently, 5 μL of the recombinant plasmid was added to competent cells of A. tumefaciens strain GV3101. The mixture was incubated on ice for 30 min, snap‐frozen in liquid nitrogen for 5 min, thawed in a 37°C water bath for 5 min, and placed back on ice for another 2 min. Subsequently, 800 μL of liquid LB medium was supplemented, and the bacteria were cultured at 28°C with shaking at 240 rpm for 3 h. The cell suspension was spread onto solid LB medium containing 20 μg ml−1 rifampicin (Rif) and 50 μg ml−1 kanamycin (Kan), followed by incubation at 28°C for two days. Single colonies were picked for colony PCR verification, and the positive amplicons were sent to Sangon Biotech for Sanger sequencing. Strains with sequences perfectly matching the target fragment were preserved as positive clones for subsequent experiments. The positive Agrobacterium was cultured at 28°C for 18 h, resuspended in infiltration buffer to OD600 = 1.2, and infiltrated into tobacco leaves to express 35S‐PmMYB‐eGFP transiently. Plants were incubated in darkness for 48 h post‐infiltration. Fluorescence signals of GFP and mCherry were observed and imaged using a TCS confocal laser scanning microscope (Leica Microsystems) with the following excitation/emission wavelengths: GFP with an excitation of 488 nm and an emission of 500–530 nm and mCherry with an excitation of 561 nm and an emission of 580–620 nm.

2.4. qRT‐PCR Analysis

The masson pine trees were collected from Ma'anshan State‐owned Forest Farm in Duyun City, Guizhou Province, China. Twenty 12‐year‐old masson pine trees with similar diameter at breast height (DBH) and tree height were selected. Holes with a diameter of 1 cm and a depth of 2 cm were drilled at 1.2 m above the ground to collect pine resin, which was used to calculate the daily pine resin yield (Shen et al. 2025). Resin yield data are provided in Table S2. Total RNA was extracted using the plant RNA extraction kit (OMEGA). First‐strand cDNA was synthesized from the extracted RNA with a reverse transcription (RT) reagent kit (TaKaRa). qRT‐PCR was performed using the SYBR qPCR Mix (GenStar) on a qTOWER3ᴳ (Analytik Jena) thermal cycler. Gene‐specific fluorescent primers for PmMYB30, PmMYB68, and PmMYB74 were designed using Premier 5.0 (Table S1). Primers used for tobacco expression analysis were adopted from a previous study (Gao, Meng, Wang, Chen, Zhou, et al. 2023). The relative expression levels of target genes were calculated using the 2–ΔCt and 2–ΔΔCt methods (Livak and Schmittgen 2001).

2.5. Transcriptional Activity Analysis

To assess transcriptional activation, truncated constructs were generated based on the conserved domains of PmMYB30, PmMYB68, and PmMYB74 via segmental cloning (Table S1). These amplicons were subsequently cloned into the pGBKT7 vector via seamless cloning. The resulting recombinant plasmids were individually transformed into the yeast Y2HGold. The transformants were plated on SD medium without Trp and incubated at 30°C for two days to select for colonies harboring the plasmids. Single colonies were subjected to colony PCR, and amplicons from clones yielding a single product were sequenced by Sangon Biotech. To evaluate transcriptional activity, confirmed positive colonies were serially diluted, spotted onto SD medium without Trp, His, and Ade supplemented with X‐α‐Gal, and incubated. The pGBKT7‐VP16 plasmid, which contains a strong activation domain, was used as a positive control in parallel assays.

2.6. Genetic Transformation of Tobacco and Phenotype Measurement

Overexpression constructs containing the coding sequences of PmMYB30, PmMYB68, and PmMYB74 under the control of the cauliflower mosaic virus 35S promoter were generated. The resulting constructs were separately introduced into GV3101 (for transformation steps refer to Section 2.3). Transgenic tobacco plants were subsequently generated via the leaf disc transformation method (Gallois and Marinho 1995). Transgenic lines were selected using hygromycin, and genomic DNA was extracted for PCR verification. For phenotypic analysis, wild‐type (WT) and homozygous transgenic plants were grown concurrently under identical environmental conditions for 35 days. Plants were cultivated in an RDN‐1000C artificial climate incubator (Dongnan Instrument) at 25°C ± 2°C under a 14 h light/10 h dark photoperiod. The built‐in plant growth LED lamps provided a photosynthetic photon flux density (PPFD) of approximately 280 μmol m−2 s−1 Subsequently, plant height, leaf length, and leaf width were measured.

2.7. SEM

For SEM, 2 cm2 sections were excised from the mid‐region of fully expanded leaves of WT, OE‐PmMYB30‐3, OE‐PmMYB30‐1, OE‐PmMYB68‐6, OE‐PmMYB68‐4, OE‐PmMYB74‐3, and OE‐PmMYB74‐2 transgenic tobacco plants. The samples were immediately fixed in primary fixative (Servicebio) at room temperature for 2 h. After fixation, they were rinsed with 0.1 M phosphate buffer (PB), then post‐fixed with 1% osmium tetroxide in the dark for 1 h and rinsed again with PB. Subsequently, dehydration of the samples was achieved through a graded ethanol series, followed by immersion in isoamyl acetate, and critical point drying was conducted with a K850 dryer (Quorum Technologies). Following this, the samples were then coated with a thin layer of gold using an MC1000 ion sputter coater (Hitachi High‐Technologies Corporation). Finally, the samples were observed and imaged with a SU8100 scanning electron microscope (Hitachi High‐Technologies Corporation). All images were captured at a consistent magnification and field of view to ensure comparability.

2.8. UPLC‐MS/MS

Leaf samples from WT and transgenic tobacco, stored at −80°C, were lyophilized using a vacuum freeze‐dryer (Scientz‐100F; Ningbo Xinzhi). The dried samples were ground into a fine, homogeneous powder with a mixer mill (MM 400, Retsch). For metabolite extraction, 50 mg of the powdered tissue was mixed with 1200 μL of ice‐cold (−20°C) 70% aqueous methanol containing internal standards (for extraction efficiency correction). The mixture was vortexed for 30 s every 30 min for six cycles in total. After thorough vortexing, the sample was centrifuged at 14,328g for 3 min, and the supernatant was collected afterward, filtered through an organic phase microporous membrane (Merck Millipore), and transferred into a vial for UPLC‐MS/MS analysis. Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 Column (1.8 μm, 2.1 mm × 100 mm) with ultrapure water containing 0.1% formic acid as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B, while the column temperature was set at 40°C, the flow rate at 0.40 mL min−1, and the injection volume at 4 μL. Metabolite separation and detection were conducted on a UPLC‐MS/MS system consisting of an LC‐30A ultra‐performance liquid chromatography unit (Shimadzu) coupled to a mass spectrometer.

2.9. Y2H Assay

For Y2H library screening, a truncated sequence of PmMYB30 without the autoactivation domain was inserted into pGBKT7 to construct the pGBKT7‐PmMYB30 vector as a bait, which was then co‐transformed with library plasmids into Y2HGold yeast cells (for transformation steps refer to Section 2.1). The transformants were plated onto SD medium without Trp, Leu, and His. Colonies that grew were then transferred to a more stringent SD medium without Trp, Leu, His, and Ade supplemented with X‐α‐Gal. Blue colonies were picked for colony PCR. Amplicons that produced a single band were sequenced, and the resulting sequences were aligned against the NCBI database to identify the interacting proteins. For one‐to‐one interaction validation, the coding sequence of the candidate protein PmRDUF1 was amplified with specific primers and cloned into the pGADT7 prey vector. The resulting pGADT7‐PmRDUF1 construct was then cotransformed with the bait plasmid pGBKT7‐PmMYB30 into Y2HGold yeast cells. Protein–protein interaction was assessed by spotting the cotransformants onto SD medium without Trp, Leu, His, and Ade containing X‐α‐Gal.

2.10. Luciferase Complementation Imaging Assay

Specific primers were used to clone PmMYB30 and PmRDUF1 into the nLUC and cLUC vectors, respectively, via seamless recombination, generating the nLUC‐PmMYB30 and cLUC‐PmRDUF1 constructs. Both constructs were introduced into GV3101. Bacterial suspensions carrying the two constructs were co‐infiltrated into leaves of tobacco. To detect the luminescence signal, a d‐luciferin potassium salt solution was applied to the infiltrated leaf areas, and images were captured using the PlantView100 (BLT).

2.11. Statistical and Correlation Analysis

The data are shown as the average ± standard deviation (SD) from a minimum of three separate biological experiments. To determine statistical significance among several groups, a one‐way ANOVA was conducted, followed by a Duncan's post hoc test, using SPSS version 21.0 (IBM Corp.). A p‐value of less than 0.05 was deemed statistically significant. Pearson correlation coefficients were calculated via bivariate correlation analysis, with significance evaluated using a two‐tailed test. Origin 2021 (OriginLab Corp.) was used to create all figures.

3. Results

3.1. Three PmMYBs Directly Bind to the PmLAS Promoter to Regulate Its Activity

Previously, it was established that the PmLAS gene plays an important role in terpenoid biosynthesis in masson pine. To identify upstream transcription factors regulating PmLAS, we performed a Y1H screening of a masson pine cDNA library using a bait construct harboring the PmLAS promoter. This screen identified three genes encoding MYB‐type transcription factors, which were designated as PmMYB30, PmMYB68, and PmMYB74 (Table S3). Phylogenetic tree analysis revealed that PmMYB30 and PmMYB74 share a relatively recent ancestral origin with the gymnosperm MYB family and are evolutionarily conserved. In contrast, PmMYB68 clustered with Prunus yedoensis MYB1R1 and Macadamia integrifolia MYB1R1 (both angiosperms), belongs to a specialized MYB1R subtype (Figure S1). The Y1H pairwise verification assay revealed successful growth of all yeast cells on SD medium without Trp, Leu, and His. Upon 3AT was added, only yeast cells from the positive control and those co‐transformed with the prey constructs (pGADT7‐PmMYB30, pGADT7‐PmMYB68, and pGADT7‐PmMYB74) and the bait construct (pHIS2‐PmLAS) survived, while the negative control did not grow (Figure 1A–C). These results indicate that PmMYB30, PmMYB68, and PmMYB74 are all capable of binding to the PmLAS promoter.

FIGURE 1.

FIGURE 1

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PmMYB30, PmMYB68, and PmMYB74 bind to the PmLAS promoter and activate its expression. (A–C) Yeast one‐hybrid assays of the PmLAS promoter with three PmMYB transcription factors (PmMYB30, PmMYB68, and PmMYB74). Combinations of pHIS2‐PmLAS with pGADT7‐PmMYB30, pGADT7‐PmMYB68, and pGADT7‐PmMYB74 served as the experimental groups, while pHIS2‐PmLAS + pGADT7 served as the control group. (D–I) Dual‐luciferase assays of PmLAS and the three PmMYB transcription factors (PmMYB30, PmMYB68, and PmMYB74) in tobacco, where pGreen‐62SK + pGreen0800‐proPmLAS‐Luc served as the control group (1), and combinations of pGreen‐0800‐proPmLAS‐Luc with pGreen‐62SK‐PmMYB30, pGreen‐62SK‐PmMYB68, and pGreen‐62SK‐PmMYB74 (2) served as the experimental groups. The symbols *, **, and *** denote statistical significance at the p < 0.05, p < 0.01, and p < 0.001 levels, respectively.

To determine the regulatory effect of these transcription factors (PmMYB30, PmMYB68, and PmMYB74) on PmLAS expression, pGreen‐62SK‐PmMYB vectors were constructed. Additionally, the PmLAS promoter was used to construct the pGreen‐0800‐proPmLAS‐luc vector, and dual‐luciferase reporter assays were performed by co‐injecting these vectors into tobacco leaves. The results indicated that, in comparison to the control group (pGreen‐62SK + pGreen‐0800‐proPmLAS‐Luc), the luminescent signals in the three experimental groups (pGreen‐62SK‐PmMYB30/PmMYB68/PmMYB74 + pGreen‐0800‐proPmLAS‐Luc) were significantly enhanced (Figure 1D–F). Enzyme activity assays indicated that the relative luciferase activity observed in the experimental groups was significantly elevated compared to that of the control group (Figure 1G–I). These results indicate that all three PmMYB transcription factors can directly bind to the PmLAS promoter and activate its expression.

3.2. PmMYB30, PmMYB68, and PmMYB74 Possess the Function of Transcription Factors

To determine the subcellular localization of PmMYB30, PmMYB68, and PmMYB74, transient expression assays of 35S‐PmMYB30‐eGFP, 35S‐PmMYB68‐eGFP, and 35S‐PmMYB74‐eGFP in tobacco leaves were performed. 35S‐eGFP (control vector) produced GFP signals throughout the entire cell. By contrast, GFP signals of 35S‐PmMYB30‐eGFP, 35S‐PmMYB68‐eGFP, and 35S‐PmMYB74‐eGFP were exclusively observed within the nucleus and colocalized with the nuclear localization marker (Figure 2A). This observation suggests that PmMYB30, PmMYB68, and PmMYB74 are nuclear proteins, aligning with their roles as transcription factors.

FIGURE 2.

FIGURE 2

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Subcellular localization and transcriptional activation assay of PmMYB30, PmMYB68, and PmMYB74. (A) Subcellular localization of PmMYB30, PmMYB68, and PmMYB74 in tobacco leaves. (B–D) Transcriptional activation assay in a yeast system. pGBKT7‐Vp16 served as the positive control and pGBKT7 as the negative control.

To examine the transcriptional activity of PmMYB30, PmMYB68, and PmMYB74, transcriptional reporter assays were performed using yeast cells. The results from the growth assays demonstrated that yeast cells transformed with constructs containing the full‐length and C‐terminal regions of all three PmMYB transcription factors exhibited normal growth on selective media and displayed α‐galactosidase activity (Figure 2B–D). These findings suggest that the C‐terminal region is crucial for the transcriptional activity of these PmMYB transcription factors.

3.3. Expression and Correlation Analysis of PmMYB30, PmMYB68, and PmMYB74 in Masson Pine With Different Resin Yields

Twenty 12‐year‐old masson pines were selected from the Ma'anshan State‐owned Forest Farm for the subsequent studies (Figure 3A). The daily resin yield was determined by measuring the weight of collected resin (Table S1). These trees were then sequentially numbered 1–20 according to their resin yields from low to high. Thereafter, the expression levels of three PmMYB genes in three tissues of masson pine with different resin yields were analyzed (Figure 3B–D). The results demonstrated that the expression of three PmMYBs in the xylem exhibited an upward trend alongside a rising resin yield, while no obvious trend was observed in needles and branches. Correlation analysis was further performed (Figure 3E), and the expression of PmMYB30, PmMYB68, and PmMYB74 in the xylem showed a significantly positive correlation with resin yield, among which PmMYB30 gave the highest correlation coefficient (r = 0.57). The expression of PmMYB74 in branches showed a negative correlation (r = −0.35) with resin yield.

FIGURE 3.

FIGURE 3

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Expression and correlation analysis of PmMYB30, PmMYB68, and PmMYB74 in three tissues (mature needles, mature branches, and trunk xylem) of 12‐year‐old masson pines with different resin yields. (A) Sampling schematic for masson pine. (B–D) Line graphs showing the expression levels of PmMYB30, PmMYB68, and PmMYB74 in three tissues of masson pine with different resin yields. Numbers 1–20 on the x‐axis represent 20 plants with resin yields from low to high. (E) Correlation heatmap between the expression levels of PmMYB30, PmMYB68, PmMYB74 and resin yield. The symbols *, **, and *** denote statistical significance at the p < 0.05, p < 0.01, and p < 0.001 levels, respectively.

3.4. Overexpression of PmMYB30, PmMYB68, and PmMYB74 Alters Tobacco Growth Phenotypes

Overexpression constructs for PmMYB30, PmMYB68, and PmMYB74 were generated and used for the genetic transformation of tobacco. For each PmMYB gene, six independent positive transgenic lines were obtained. qRT‐PCR analysis confirmed the expression of the transgenes (Figure 4A–C). The high‐expression lines designated as OE‐PmMYB30‐3, OE‐PmMYB30‐1, OE‐PmMYB68‐6, OE‐PmMYB68‐4, OE‐PmMYB74‐3, and OE‐PmMYB74‐2 were selected for further phenotypic characterization. Compared to the WT, the phenotypes of transgenic lines were manifested as distinct growth inhibition (Figure 4D–G). All three overexpression lines exhibited significantly shorter leaves. Furthermore, the PmMYB30 overexpression lines (OE‐PmMYB30‐3 and OE‐PmMYB30‐1) and PmMYB74 overexpression lines (OE‐PmMYB74‐3 and OE‐PmMYB74‐2) displayed a significant reduction in both plant height and leaf width. Overall, these results revealed that overexpression of PmMYB30, PmMYB68, and PmMYB74 influences tobacco growth, with the most pronounced inhibitory effect on plant height, leaf length, and leaf width observed in the PmMYB30 overexpression lines.

FIGURE 4.

FIGURE 4

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Analysis of growth parameters in transgenic tobacco lines overexpressing PmMYB30, PmMYB68, and PmMYB74. (A–C) The relative expression levels of PmMYB30, PmMYB68, and PmMYB74 in transgenic lines were determined by qRT‐PCR. (D) Phenotypic comparison of WT and transgenic plants. (E–G) Quantification of plant height, leaf length, and leaf width. One‐way ANOVA and Duncan's post hoc test were used to calculate significance, and different letters indicate significance at p < 0.05.

Leaf epidermal glandular trichomes were next examined by SEM (Figure 5A–G). Quantitative analysis of trichome density within a defined field of view showed that all three PmMYB overexpressing lines exhibited a significantly higher number of glandular trichomes compared to the WT (Figure 5H), among which PmMYB30 overexpression lines displayed the greatest increase in glandular trichome density.

FIGURE 5.

FIGURE 5

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Analysis of leaf glandular trichome numbers in WT and OE‐PmMYB30‐3, OE‐PmMYB68‐6, and OE‐PmMYB74‐3 transgenic tobacco lines. (A–D) Scanning electron microscopy (SEM) images of leaf glandular trichomes from WT and OE‐PmMYB30‐3, OE‐PmMYB68‐6, and OE‐PmMYB74‐3 transgenic plants. (E) Quantification of glandular trichome numbers per unit leaf area in WT and transgenic lines. Representative SEM images are displayed, and quantitative data were obtained from all six independent transgenic lines. One‐way ANOVA and Duncan's post hoc test were used to calculate significance, and different letters indicate significance at p < 0.05.

3.5. Overexpression of PmMYB30, PmMYB68, and PmMYB74 Modulates Terpenoid Biosynthesis in Tobacco

Subsequent analyses were conducted on the expression profiles of pivotal genes implicated in terpenoid biosynthesis (Figure 6A–F). This included the rate‐limiting gene of the MVA pathway (HMGR), two key genes of the MEP pathway (DXS and DXR), and the backbone synthesis genes for mono‐, sesqui‐, and diterpenoids (GPPS, FPPS, and GGPPS, respectively). The expression of DXS was markedly upregulated in all three PmMYB‐overexpressing lines in comparison with that of the WT, with the highest level in PmMYB30 overexpression lines (OE‐PmMYB30‐3 and OE‐PmMYB30‐1). In contrast, DXR expression was significantly downregulated in OE‐PmMYB68‐6, OE‐PmMYB68‐4, and OE‐PmMYB74‐3. Both of HMGR and FPPS were significantly downregulated in all three transgenic lines. Furthermore, GPPS expression increased significantly only in PmMYB30 overexpression lines, while GGPPS expression was significantly upregulated in all three PmMYB transgenic lines.

FIGURE 6.

FIGURE 6

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Expression of key terpene synthase genes in WT and transgenic tobacco lines (OE‐PmMYB30‐3, OE‐PmMYB68‐6, and OE‐PmMYB74‐3). (A–F) The expression levels of DXS, DXR, HMGR, GPPS, FPPS, and GGPPS were detected in wild‐type and transgenic tobacco lines. One‐way ANOVA and Duncan's post hoc test were used to calculate significance, and different letters indicate significance at p < 0.05.

Metabolite profiling of WT and transgenic plants was subsequently performed. The total terpenoid content increased in PmMYB30 overexpressing lines (OE‐PmMYB30‐3, 2.90%; OE‐PmMYB30‐1, 2.92%) and PmMYB74 overexpressing lines (OE‐PmMYB74‐3, 2.83%; OE‐PmMYB74‐2, 2.79%) compared to WT (2.39%), whereas no significant difference was observed in PmMYB68‐overexpressing lines (OE‐PmMYB68‐6, 2.32%; OE‐PmMYB68‐4, 2.38%; Figure 7A). Analysis of individual terpenoid classes revealed substantial alterations (Figure 7B). The relative content of diterpenoids was markedly elevated in transgenic lines. Compared with 15.32% in the WT, the proportions of diterpenoids reached 39.56% in OE‐PmMYB30‐3, 40.12% in OE‐PmMYB30‐1, 26.60% in OE‐PmMYB68‐6, 27.42% in OE‐PmMYB68‐4, 38.43% in OE‐PmMYB74‐3, and 36.51% in OE‐PmMYB74‐2. This shift coincided with a decreased proportion of tetraterpenoids. The relative abundance of triterpenoids remained stable, while the proportions of monoterpenes and sesquiterpenes decreased. Specifically, within monoterpenes, levels of major compounds such as menthone and geraniol were reduced, most notably in PmMYB30 overexpressing lines and PmMYB74 overexpressing lines (Figure 7C). For sesquiterpenes, α‐farnesene decreased specifically in PmMYB74 overexpressing lines, and farnesol derivatives declined in all transgenic lines (Figure 7D). In diterpenoids, α‐Cembratriene‐4,6‐diol accumulated in all lines, while cembratriene‐4‐ol increased only in PmMYB74 overexpressing lines (Figure 7E). Among triterpenes, the metabolic profile shifted, with β‐amyrin decreasing and cycloartenol increasing (Figure 7F). Finally, the tetraterpene all‐trans‐neoxanthin was significantly reduced in all transgenic plants (Figure 7G). Previous results indicated that the expression of PmMYB30 exhibited the strongest correlation with the resin yield. Furthermore, its overexpression in transgenic tobacco, which exhibited the most pronounced reduction in growth, also displayed the greatest increase in trichome density, the highest proportion of diterpenoids, and an elevated total terpenoid proportion.

FIGURE 7.

FIGURE 7

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Analysis of metabolite profiles in OE‐PmMYB30‐3, OE‐PmMYB68‐6, and OE‐PmMYB74‐3 transgenic tobacco lines.

3.6. PmRDUF1 Interacts With PmMYB30 and Inhibits the Activation of PmLAS Expression

To further investigate the regulatory network of PmMYB30 in terpenoid biosynthesis, we carried out Y2H library screening using the masson pine cDNA library. The bait vector pGBKT7‐PmMYB30 was constructed by cloning a truncated PmMYB30 fragment without the autoactivation domain into pGBKT7. As a result, one E3 ubiquitin ligase was identified, which was designated as PmRDUF1. Phylogenetic analysis revealed that the protein encoded by this gene belongs to the RING‐Type E3 Ubiquitin Ligase family. It shares high homology with RDUF1 proteins from Gossypium hirsutum and Glycine max , indicating a close evolutionary relationship (Figure S2). To verify this interaction, a direct Y2H assay was performed. The results showed that all yeast cells grew normally on SD medium without Trp and Leu. In contrast, on SD medium without Trp, Leu, His, and Ade with X‐α‐Gal, only yeast cells of the positive control and the experimental group (pGBKT7‐PmMYB30 + pGADT7‐PmRDUF1) grew normally and exhibited α‐galactosidase activity (Figure 8A). To further confirm the interaction, nLUC‐PmMYB30 and cLUC‐PmRDUF1 vectors were constructed, which were co‐infiltrated into tobacco leaves. Strong luminescence signals were observed in leaves co‐expressing nLUC‐PmMYB30 and cLUC‐PmRDUF1 (Figure 8B). These results demonstrated that the PmMYB30 protein interacts with the PmRDUF1 protein.

FIGURE 8.

FIGURE 8

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Interaction between the PmRDUF1 and PmMYB30 proteins. (A) Yeast two‐hybrid verification of PmRDUF1 and PmMYB30. (B) Luciferase complementation assay of PmRDUF1 and PmMYB30. (1) nLUC‐empty vector + cLUC‐empty vector; (2) nLUC‐empty vector + cLUC‐PmRDUF1; (3) nLUC‐PmMYB30 + cLUC‐empty vector; (4) nLUC‐PmMYB30 + cLUC‐PmRDUF1. (C‐D) Dual‐luciferase reporter assay of PmRDUF1, PmMYB30, and the PmLAS gene promoter. (1) pGreen‐62SK + pGreen‐0800‐proPmLAS‐Luc; (2) pGreen‐62SK‐PmMYB30 + pGreen‐0800‐proPmLAS‐Luc; (3) pGreen‐62SK‐PmRDUF1 + pGreen‐0800‐proPmLAS‐Luc; (4) pGreen‐62SK‐PmMYB30 + pGreen‐62SK‐PmRDUF1 + pGreen‐0800‐proPmLAS‐Luc. One‐way ANOVA and Duncan's post hoc test were used to calculate significance, and different letters indicate significance at p < 0.05.

To investigate the effect of the PmRDUF1‐PmMYB interaction on PmLAS expression, a dual‐luciferase reporter assay was performed. The pGreen‐0800‐proPmLAS‐Luc, pGreen‐62SK‐PmMYB30, and pGreen‐62SK‐PmRDUF1 vectors were co‐infiltrated into tobacco leaves. Consistent with previous results, strong luminescence signals were detected in the combination of pGreen‐0800‐proPmLAS‐Luc + pGreen‐62SK‐PmMYB30. In contrast, the luminescence signals in the co‐infiltrated group of pGreen‐0800‐proPmLAS‐Luc + pGreen‐62SK‐PmMYB30 + pGreen‐62SK‐PmRDUF1 were comparable to those of the control group (the pGreen‐0800‐proPmLAS‐Luc + empty pGreen‐62SK vector; Figure 8C). The results of LUC activity assays were consistent with the imaging results (Figure 8D). Taken together, these findings confirm that PmRDUF1 interacts with PmMYB30 and inhibits its ability to activate the transcription of PmLAS.

4. Discussion

4.1. Involvement of PmMYB Transcription Factors in Masson Pine Terpenoid Biosynthesis Through PmLAS Regulation

The PmLAS gene, encoding a key diterpene synthase, was previously shown to be crucial for levopimaric acid biosynthesis and overall resin yield in masson pine (Shen et al. 2025). However, the upstream transcriptional regulators controlling its expression have remained unknown so far. In the present study, we identified three PmMYB transcription factors, that is, PmMYB30, PmMYB68, and PmMYB74, which directly bind to and activate the PmLAS. This finding aligns with numerous studies across plant species that have established MYB transcription factors as regulators of terpenoid biosynthesis, often by modulating the expression of TPS genes. For instance, in rose ( Rosa rugosa ), MYB5 coordinates the biosynthesis of sesquiterpenes and proanthocyanidins by regulating the expression of TPS31 and ANR (Li et al. 2022), while in mountain pepper ( Litsea cubeba ), LcMYB44 exerts a negative regulatory effect on citral biosynthesis through its direct interaction with the promoter region of an alcohol dehydrogenase (ADH) gene (Zhao et al. 2024). Similarly, in Lilium, LiMYB1, LiMYB305, and LiMYB330, coordinately regulate terpene biosynthesis by activating the LiTPS2 promoter as well as via interacting with each other (Guo et al. 2023). In masson pine, PmMYB4 may interact with PmbZIP2 to co‐regulate downstream TPS and cytochrome P450 (CYP450), thereby participating in the regulation of resin biosynthesis (Chen et al. 2025). Furthermore, our data revealed that the expression levels of three PmMYB genes showed a significantly positive correlation with resin yield in masson pine, with PmMYB30 exhibiting the strongest correlation. These findings indicate that PmMYB30, PmMYB68, and PmMYB74 are involved in terpenoid biosynthesis by regulating the expression of the PmLAS.

4.2. Effects of PmMYBs Overexpression on Tobacco Growth and Terpenoid Biosynthesis

The heterologous overexpression of the masson pine genes PmMYB30, PmMYB68, and PmMYB74 in tobacco was performed in this study. The most pronounced phenotypic alterations were observed in the PmMYB30 overexpressing and PmMYB74 overexpressing lines, which exhibited significant growth inhibition, characterized by reduced plant height, leaf length, and leaf width. Concurrently, all transgenic lines also displayed a marked increase in leaf glandular trichome quantity, which was most prominent in the PmMYB30 overexpressing line. Glandular trichomes are the first chemical defense line against various threats and also key organs for the biosynthesis of various secondary metabolites (Schilmiller et al. 2008; Schuurink and Tissier 2020). Previous studies have shown similar phenotypes. For instance, heterologous expression of the tea plant‐derived α‐farnesene synthase (AFS) gene in tobacco led to the enhanced glandular trichome density alongside significant changes in the terpenoid profile of the plants, including levels of compounds like nerolidol (Dao et al. 2025). Similarly, heterologous expression of Lavandula angustifolia LaMYC4 in tobacco enhanced both sesquiterpenoid accumulation and glandular trichome density (Dong et al. 2022). Likewise, overexpression of Chrysanthemum indicum CiMYC2, CiGPS, and CiFPS led to a coordinated phenotype of dwarfism, increased glandular trichome number, and elevated terpenoid levels (Gao, Meng, Wang, Chen, He, et al. 2023; Gao, Meng, Wang, Chen, Zhou, et al. 2023).

The biosynthesis of terpenoids relies on two distinct pathways: the MVA pathway, which provides precursors for sesquiterpenoids, triterpenoids, and sterols, and the methylerythritol MEP pathway, which supplies precursors for monoterpenoids, diterpenoids, and tetraterpenoids (Chappell 2002; Rodríguez‐Concepción and Boronat 2015). Supplementing intermediates of either the MVA or MEP pathway, or using chemical inhibitors to suppress one of them, can enhance the extent of precursor exchange, thereby altering precursor allocation (Kasahara et al. 2002; Bick and Lange 2003; Hemmerlin et al. 2003; Rodríguez‐Concepción et al. 2004; Paetzold et al. 2010). In our study, the transcript level of DXS, a key gene in the MEP pathway, was significantly upregulated, whereas that of HMGR, which encodes the rate‐limiting enzyme of the MVA pathway, was suppressed. These transcriptional changes suggest a preferential allocation of precursors toward the MEP pathway, a conclusion supported by the observed shifts in the metabolite profiles. Specifically, the proportion of diterpenoids increased significantly in the transgenic plants, reaching up to 40% in the PmMYB30 overexpressing lines, and was accompanied by the accumulation of major diterpenes like α‐cembratriene‐4,6‐diol. Conversely, the relative contents of sesquiterpenoids decreased. In G. biloba , the expression of GbDXS is correlated with the accumulation of the diterpenoid ginkgolides (Gong et al. 2006). In Solanum lycopersicum , downregulation of SlDXS2 led to a decrease in the monoterpene β‐phellandrene within trichomes, accompanied by an increase in the accumulation of two sesquiterpenes (Paetzold et al. 2010). In summary, these results suggest that PmMYB transcription factors activate the MEP pathway, promote diterpenoid biosynthesis, and induce glandular trichome development, thereby initiating the chemical defense system in plants. Enhanced defensive metabolism redirects and re‐orchestrates metabolic precursors toward secondary metabolism rather than vegetative growth, ultimately leading to a coordinated phenotype featuring elevated terpenoid levels (especially diterpenoids), increased glandular trichome density, and suppressed plant growth. These three phenotypic changes are not independent events, but integrated consequences of prioritized defense metabolism under the growth–defense trade‐off in plants. Our findings elucidated the transcriptional mechanism underlying these metabolic shifts, demonstrating that terpenoid biosynthesis in masson pine is governed by a PmMYB‐PmLAS regulatory module. Particularly, three PmMYB transcription factors, that is, PmMYB30, PmMYB68, and PmMYB74, directly regulate the expression of the diterpene synthase gene PmLAS, thereby controlling diterpenoid production.

4.3. The Regulatory Role of the PmRDUF1‐PmMYB30‐PmLAS Module in Terpenoid Biosynthesis

By specifically recognizing and ubiquitinating target proteins, E3 ubiquitin ligases establish the specificity of the ubiquitin‐proteasome system, representing a pivotal mechanism in plant adaptation to abiotic stress (Finley and Varshavsky 1985; Hershko and Ciechanover 1998; Chen and Hellmann 2013; Shu and Yang 2017). Building upon the discovery that PmMYB30 is a direct transcriptional activator of PmLAS, the present study uncovered an additional layer of regulation through protein–protein interactions. Y2H pairwise assays and luciferase complementation imaging assays confirmed the physical interaction between PmMYB30 and PmRDUF1 proteins. Furthermore, dual‐luciferase reporter assays demonstrated that PmRDUF1 interacts with PmMYB30 to suppress its activation of PmLAS expression. This mode of regulation is not unprecedented: for instance, in Arabidopsis thaliana , the COP1/SPA E3 ubiquitin ligase complex modulates the protein stability of the MYB transcription factors PAP1 and PAP2, thereby influencing anthocyanin accumulation (Maier et al. 2013). Similarly, in Chrysanthemum (chrysanthemum × morifolium Ramat.), CmPUB15 targets CmMYB73 for degradation via the 26S proteasome pathway, thereby relieving the repression exerted by CmMYB73 on anthocyanin biosynthesis (Geng et al. 2025). Based on the current evidence and previous studies, we speculate that PmRDUF1 targets PmMYB30 for ubiquitination and subsequent proteasomal degradation, which in turn suppresses the PmMYB30‐mediated activation of PmLAS. Therefore, the formation of the PmRDUF1‐PmMYB30‐PmLAS tripartite module reveals a complex, multilayered regulatory circuit for diterpenoid biosynthesis in masson pine. This architecture allows for the dynamic and potentially signal‐responsive fine‐tuning of resin yields. Although the current study has yielded important insights into the functional role of PmMYB transcription factors and the PmRDUF1‐PmMYB30‐PmLAS regulatory module involved in terpene biosynthesis in masson pine, several limitations still remain unresolved. Firstly, due to the lack of a transformation system in masson pine, functional characterization of PmMYB30, PmMYB68, PmMYB74, and PmRDUF1 was mainly performed via heterologous overexpression in tobacco, which cannot fully recapitulate their authentic regulatory environment in native masson pine. Secondly, the regulatory mechanism of the PmRDUF1‐PmMYB30‐PmLAS module needs further elucidation; for instance, the specific ubiquitination sites of PmMYB30 targeted by PmRDUF1 remain undetermined, and direct evidence supporting the protein stability of PmMYB30 is lacking. Therefore, the present study provides a theoretical basis for the molecular improvement of masson pine, and marker‐assisted selection (MAS) based on the polymorphic loci of this gene can thereby accelerate the breeding process of high‐resin‐yield masson pine varieties since PmMYB30 exhibits a significant positive correlation with resin yield.

5. Conclusions

This study systematically deciphered the multilayered regulatory network controlling terpenoid biosynthesis in masson pine. We discovered that PmMYB30, PmMYB68, and PmMYB74 function as direct transcriptional activators of the key diterpene synthase gene PmLAS. Their expression levels positively correlated with resin yield, and their heterologous overexpression in tobacco led to the significant enrichment of diterpenoids, thereby establishing a foundational PmMYB‐PmLAS regulatory module. Furthermore, we expanded this module by identifying an interaction between the E3 ubiquitin ligase PmRDUF1 and PmMYB30, which likely promotes PmMYB30 degradation via the ubiquitin‐proteasome pathway, thus forming an intricate PmRDUF1‐PmMYB30‐PmLAS regulatory circuit (Figure 9). Collectively, these findings delineate a sophisticated regulatory network and offer novel insights for modulating terpenoid production in masson pine.

FIGURE 9.

FIGURE 9

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Schematic diagram of the PmRDUF1‐PmMYB30‐PmLAS regulatory module in the terpenoid biosynthesis of masson pine. The expression of the PmLAS gene significantly influences levopimaric acid content and, consequently, resin yield. Three PmMYB transcription factors (PmMYB30, PmMYB68, and PmMYB74) were found to bind to and activate the PmLAS promoter. Furthermore, the E3 ubiquitin ligase PmRDUF1 interacts with PmMYB30. This interaction inhibits the ability of PmMYB30 to activate PmLAS expression, thereby forming a PmRDUF1‐PmMYB30‐PmLAS regulatory module that participates in terpenoid biosynthesis in masson pine.

Author Contributions

Luonan Shen: conceptualization, data curation, formal analysis, methodology, software, resources, visualization, writing – original draft, writing – review and editing. Sulin Wen: methodology, software, writing – review and editing. Lin Deng: data curation, methodology, resources. Kui Zhou: software, resources, writing – review and editing. Kun Yang: conceptualization, software, methodology. Guang Qiao: conceptualization, resources, methodology. Yi Min: data curation, formal analysis. Lei Peng: methodology, writing – review and editing. Chaojin Wei: data curation, resources. Zhengdong Cai: data curation, resources. Xiaopeng Wen: conceptualization, funding acquisition, investigation, project administration, resources, supervision, validation, writing – review and editing.

Funding

This work was financially supported by the grants from Guizhou Provincial Science and Technology Projects (QKHZHYD [2024] 044) and Guizhou Provincial Major Scientific and Technological Program (No. 2024010).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Phylogenetic analysis of PmMYB transcription factors.

Figure S2: Phylogenetic analysis of PmRDUF1 transcription factors.

Table S1: Primers.

Table S2: Parameters of 12‐year‐old masson pine.

Table S3: Results of yeast one‐hybrid library screening.

Table S4: Relative contents of the main terpenoid components in transgenic tobacco.

PPL-178-e71059-s001.pdf (401.1KB, pdf)

Acknowledgments

During the preparation of this work, the authors did not use any generative AI or AI‐assisted technologies.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Phylogenetic analysis of PmMYB transcription factors.

Figure S2: Phylogenetic analysis of PmRDUF1 transcription factors.

Table S1: Primers.

Table S2: Parameters of 12‐year‐old masson pine.

Table S3: Results of yeast one‐hybrid library screening.

Table S4: Relative contents of the main terpenoid components in transgenic tobacco.

PPL-178-e71059-s001.pdf (401.1KB, pdf)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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